Here we report the discovery and isolation of erythromycin E (2) from the culture broth of Nocardia brasiliensis IFM 0466. The strain of N. brasiliensis IFM 0466, an isolate from nocardiosis patient, is kept in the culture collection of the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University and was used in this study. Isolation of erythromycins from a 96 hour-culture broth (20-liters) was done by chromatography with a Diaion HP-20 column (3×30 cm) and elution with MeOH:H2O (4:1 and 100:0). The combined fractions active against Bacillus subtilis PCI 219 were further purified by preparative reverse-phase HPLC (Soken pack C18 Soken Co., Ltd., 2×30 cm, flow rate; 30 ml/minute, MeCN:50 mM phosphate buffer pH 6.5=38:72). At the end of the purification process 2 was obtained in a yield of 65 mg as a pure compound in addition to coproduced erythromycin A (Fig. 1) and 8,9-anhydropseudo-erythromycin A-6,9-hemiketal4). The structure of 2 from N. brasiliensis IFM 0466 was readily inferred from mass spectrometry and one- and two-dimensional NMR measurements. The electrospray mass spectrum of 2 displayed m/z 748.1 ([M+H]+). Collision-induced dissociation (ESI-CID-MS/MS) of m/z 748 with argon gas afforded diagnostic fragment ions such as m/z 574.8 ion ([monoglycosylated aglycone, -O, -desosamine, +H]+) and m/z 157.9 ([desosamine-O]~). HRFAB-MS suggested the chemical formula C37H65NO14 for 2 ([M+H]+ m/z 748.4562 found, calcd. 748.4483). In addition, the same diagnostic fragments were observed as found for ESI-CID-MS/MS. The 1H and 13C NMR spectra of 2 proved unambiguously the identity of the Nocardia metabolite 2 as erythromycin E3). Additional evidence was provided by the HSQC, TOCSY, NOESY and HMBC NMR spectra. Diagnostic C, H long-range couplings in the HMBC spectrum of 2 confirmed the ortho-carboxylic acid ester structure, its unique structural feature. Coproduced compounds 1 and 8,9-anhydro-pseudoerythromycin A-6,9-hemi-ketal were identified readily via their MS spectra (ESI-CID-MS/MS) and an NMR spectroscopic investigation4). In the crude mixture of erythromycins 1, 2 and 8,9-anhydro-pseudo-erythromycin A-6,9-hemiketal isolated from N. brasiliensis IFM 0466, the portion of erythromycin E (2) was estimated to be 64 % from the relative intensities of the [M+H]+ ions (ESI-MS), which were 1, 2.78 and 0.57 for m/z 734, 748 and 716, respectively. N. brasiliensis IFM 0466 was thus found to be a new producer of erythromycin E rendering this antibiotic available for normal fermentation processes which could supply this unusual structure for semisynthetic modifications. Only one case of the production of erythromycins from Nocardia (Nocardia sp. ATCC59045) has been reported5). However, the present Nocardia strain was easily differentiated from strain ATCC59045, which uses nitrogen or carbon sources such as adenine, xanthine and starch. Therefore, we believe that this is also the first isolation of erythromycins from pathogenic N. brasiliensis.