A new C47 acetylenic acid, nepheliosyne A [1], has been isolated from the Okinawan marine sponge Xestospongia sp. and its structure elucidated on the basis of spectroscopic data. Marine sponges of the genera Petrosia or Xestospongia (family Nepheliospongiidae) frequently afford polyacetylene metabolites (1) and most of them exhibit various bioactivities such as antifungal, cytotoxic, or antiviral activity. During our studies on bioactive substances from Okinawan marine organisms (2), we recently investigated extracts of the sponge of the genus Xestospongia and isolated three new cytotoxic and antimicrobial C19 sterols containing a cyclopropane moiety (3). Further examination of the extracts of this sponge resulted in the isolation of a new acetylenic acid metabolite of high molecular weight, nepheliosyne A [1], which proved to possess a closely related structure to petrosolic acid, a C44 oxooctahydroxytrienetetraynoic carboxylic acid quite recently isolated from a Red Sea sponge Petrosia sp. (4). The sponge Xestospongia sp. was collected off Ishigaki Island, Okinawa, and kept frozen until used. The MeOH extract of the sponge was partitioned between EtOAc and H2O. The EtOAc-soluble fraction was subjected to Si gel cc followed by C18 cc to give nepheliosyne A (1, 0.01%, wet wt) as a colorless, amorphous solid. The molecular formula of 1 was deduced as C47H70O11 by its hrfabms data [m/z 833.4766, (M+Na)+, Δ -4.0 mmu]. The ¹H- and ¹³C-nmr spectra of 1 revealed signals due to a ketone, three disubstituted double bonds, eight oxymethines, and 23 sp³ methylenes, but no methyl groups. The presence of a carboxylic acid group was suggested by the ir absorption band of 1 at 1700 cm⁻¹ as well as the fact that a methyl ester was obtained by treatment of 1 with CH₂N₂. Interpretation of the ¹H- and ¹³C-nmr data (Table 1) including ¹H-¹H COSY, HOHAHA (5), HSQC (6), and HMBC (7) nmr spectra indicated the presence of partial structures. The HMBC spectrum showed correlations due to H₂-8*/C-9* and H-10*/C-9*, and the ¹³C-nmr chemical shift of C-10 (δ 78.0) implied that this oxymethine carbon is not at a β-position but rather an α-position to a ketone carbonyl (C-9). For the partial structure C, the ¹H-¹H COSY nmr spectrum revealed proton-coupling networks: from H₂-25 to H₂-28, from H₂-30 to H-31, and from H-37 to H-45. The HMBC spectrum afforded correlations for H-27/C-29, H₂-28/C-29, H₂-28/C-30, and H-31/C-30. The ¹³C-nmr chemical shifts for C-31 (δ 62.63) and C-37 (δ 62.59) suggested these oxymethines are adjacent to acetylenic carbons (δ>80), while C-34 (δ 52.3) was inferred between two sp carbons. H-34 showed long-range coupling through a triple bond (triplet, J=1.7 Hz) to H-31 and H-37. Allylic methylenes of C-25/C-28 implied Z-configuration for Δ²⁶,²⁷, while C-38/C-41 suggested E-configuration for ³⁹E, and Δ⁴³,⁴⁴ was E (J=15.0 Hz). From COSY/HOHAHA, proton connectivities from H-4 to H₂-7 were clear. No branched substituents (except hydroxyls) meant the acetylenic bond at C-2/C-3 connects to the carboxylic acid (C-1, terminal). C-4 (δ 59.6) was adjacent to C-3 (sp carbon), forming partial structure A. Linear connection of partial structures gave the full structure of nepheliosyne A [1], a homologue of petrosolic acid (three fewer CH₂ units). Nepheliosyne A [1] is the highest molecular weight polyacetylene carboxylic acid from marine sponges and the first C47 polyacetylene natural product. Cytotoxicity was weak: 40% inhibition against lymphoma L-1210 and 14% against human epidermoid carcinoma KB cells at 20 μg/cm³ in vitro.