Rimocidin is an antifungal antibiotic isolated from the culture of Streptomyces rimosus. The partial structure including its aglycone moiety was postulated by COPE et al. Data from our laboratory prompted us to revise the structure of rimocidin aglycone and postulate the complete structure of the antibiotic. Through IR spectroscopy, mass spectrometry, potentiometric titration (of N-acetyloctahydrorimocidin to confirm one carboxyl group), chemical reactions (e.g., reaction with diazomethane to form methyl esters, treatment with dilute HCl in methanol, treatment with 1 N NaOH to form pentaenal II, preparation of permethylated derivatives) and spectroscopic analysis of derivatives (e.g., O-acetylmethoxime III of II, permethylated tetradecahydro derivatives IVa and IVb, methyl ester of N-acetyldimethoximerimocidin V and its peracetylated VI and persililated VII derivatives), we determined the attachment of the mycosamine moiety (allylic position to the chromophore, similar to known polyene macrolides), the substituted oxygen function at C17, and the carbon skeleton and positions of oxygen functions. The molecular structure was confirmed by mass spectra of V, VI, and VII. The 2,3,4-triacetylmycosamine VIII derived from VI showed identical mass and NMR spectra to that from nystatin, confirming the ring size and conformation of the aminosugar moiety.