Blasticidin S is an effective fungicide against rice blast disease in Japan. As part of its biosynthetic studies, we sought intermediates in the mother liquor from blasticidin S crystallization. A metabolite named blasticidin H (H for hydrated), which shows UV absorption characteristic of 1-substituted cytosine, was isolated. Isolation involved adsorption on active carbon, elution with 60% aqueous acetone, chromatography on Dowex 50 X-2 and Sephadex G-15, and purification as dihydrochloride. Physicochemical properties (melting point, elemental analysis, pKa values, UV spectra) suggested a close structural relationship to blasticidin S. ¹H-NMR spectroscopy revealed that blasticidin H contains an unsubstituted amino group in the blastidic acid moiety and lacks the two olefinic protons present in blasticidin S. Acid degradation yielded blastidic acid (identified by comparison with an authentic sample) and a novel nucleoside, pentopyranamine D. Structural elucidation of pentopyranamine D using ¹H-NMR (including spin decoupling), potentiometric titration, alkaline treatment of blasticidin H to form cytomycin H, and acetylation experiments confirmed its structure as 1-(4-amino-3,4-dideoxy-β-D-ribohexopyranosyluronic acid)-cytosine. Blasticidin H was thus determined to be 1-(4-blastidylamino-3,4-dideoxy-β-D-ribohexopyranosyluronic acid)-cytosine. This structure indicates that blasticidin H is a direct precursor of blasticidin S, as only dehydration is required to form blasticidin S. Blasticidin H exhibits slight activity against Piricularia oryzae (the rice blast pathogen) at high concentrations, suggesting that the double bond in the sugar moiety of blasticidin S is critical for its biological activity.