Reductive conversion of several anthracycline glycosides to their 7-deoxyaglycones occurs during their microaerophilic incubation with strains of Aeromonas hydrophila, Citrobacter freundii, and Escherichia coli. Further, extracts of microaerophilically grown A. hydrophilia catalyze DPNH-dependent reductive conversion of the same compounds. Anthracycline substrates cleaved by both whole cells and by the cell-free system include steffimycin, steffimycin B, nogalamycin, cinerubin A, and daunomycin. Investigation of glycoside cleavage as a function of both time and anthracycline concentration demonstrated the superiority of A. hydrophila over C. freundii and E. coli in regard to reaction rate and efficiency of conversion. Interestingly, some degree of anaerobicity was required for glycoside cleavage by all three organisms. Evidence supporting 7-deoxyaglycone formation via direct reductive cleavage, as opposed to a multienzyme-catalyzed process involving hydrolysis followed by dehydration and reduction, includes the following. Equilibrium mixtures of glycoside substrate and 7-deoxyaglycone product prepared using both whole cells and their extracts display no anthracycline hydrolysis products. Further, authentic steffimycinone (aglycone), the expected product of hydrolytic sugar cleavage of steffimycin, was shown to be converted to 7-deoxysteffimycinone (7-deoxyaglycone) at a rate slower than steffimycin. These data indicate that, if steffimycinone were present as an unbound metabolic intermediate, it should have been visible in the equilibrium mixture, but none was detected.