The Western Indian Ocean sponge Phakellia carteri was found to contain two isomeric (at the photo-Trp indole ring juncture) cyclo-heptapeptides named phakellistatin 3 (3) and isophakellistatin 3 (4). Our earlier evaluations of marine sponges in the genus Phakellia for cell growth inhibitory substances led to isolation of phakellistatin 1 (1) from a Western Pacific (Micronesia) Phakellia sp. and phakellistatin 2 (2) from the Eastern Indian Ocean (Republic of the Comoros) Phakellia carteri. We now report that further bioassay-guided (murine P388 lymphocytic leukemia cell line) separation of P. carteri cytotoxic constituents has resulted in the discovery of phakellistatin 3 (3), a cyclic peptide with significant P388 (ED50 0.33 μg/mL) activity. Phakellistatin 3 represents a new type of cyclo-heptapeptide containing an amino acid unit apparently derived from a photooxidation product of tryptophan. The cis-ring juncture isomer of this modified tryptophan unit was found in an otherwise identical new cyclo-heptapeptide designated isophakellistatin 3 (4). The dichloromethane-soluble fraction obtained from a 250-kg (wet wt) 1987 collection of P. carteri (order Axinellida, class Demospongiae) which was used to obtain phakellistatin 2 by a series of solvent partitioning, gel permeation, and partition chromatographic (Sephadex LH-20) procedures was further separated (P388 bioassay-guided). Continued column chromatographic separations on Sephadex LH-20 and purification by reversed-phase HPLC afforded phakellistatin 3 (3, 4.6 mg, 2.0 × 10^-6% yield) as an amorphous powder and isophakellistatin 3 (4, 30 mg, 1.3 × 10^-5% yield) as crystals from acetone. Comprehensive high-field (400- and 500-MHz) 2D-NMR analyses of cyclo-heptapeptides 3 and 4 employing APT, 1H,1H-COSY, HMQC, and HMBC techniques revealed spin systems corresponding to Gly, Leu, Pro (two units), Phe, Thr, and a Trp-like amino acid unit (3a-hydroxypyrroloindole). Sequence analyses by MS/MS techniques indicated peptides 3 and 4 differed only by stereochemistry. A specimen of isophakellistatin 3 (4) suitable for X-ray crystal structure determination crystallized from acetone; the structure was solved by direct methods, revealing association with one acetone and one water molecule. Based on the crystal structure of isophakellistatin 3, NMR NOESY experiments allowed assignment of structure 3 to phakellistatin 3, consistent with tryptophan photooxidation products (cis- and trans-photo-Trp). To our knowledge, cyclic-heptapeptides 3 and 4 represent the first examples of photo-Trp serving as a natural peptide unit. The inhibitory effect of the trans-ring juncture (peptide 3) on P388 cell growth versus lack of activity in the cis-isomer (peptide 4) is noteworthy. Extended antineoplastic evaluations of phakellistatin 3 are underway.