The biosynthetic pathway of ganefromycin alpha (8) was investigated using blocked mutants of Streptomyces lydicus spp. tanzanius, the ganefromycin alpha producer, in conjunction with bioconversion experiments with the products of these mutants. Compounds 1-7 and 9-15, which are structurally related to ganefromycin alpha, were produced by cultures of secretor mutants showing blockage or diminished production of 8. These compounds were isolated, characterized, and subjected to bioconversion experiments using converter and other secretor mutants. Some of the compounds were shown to be products resulting from blocks at various stages in the biosynthetics pathway beyond construction of the polyketide skeleton (e.g., furan ring closure, hydroxylation, glycosylation, etc.). Other compounds were not bioconverted by biosynthetically capable mutants and were deemed shunt products. A mutant's metabolites and bioconverting ability were used to surmise the location of the block. A picture of the later sequence of events leading to the synthesis of ganefromycin alpha emerged, including C23-hydroxylation, C13a-O-methylation, C21a-hydroxylation, and C21a-O-glycosylation. Some of the later steps in the biosynthetic pathway for ganefromycin alpha are proposed.