Midecamycins (SF-837) are a series of 16-membered macrolide antibiotics produced by Streptomyces mycarofaciens, classified according to structural differences at the 9-position of the lactone ring and acyl groups at the 4-position of the mycarose moiety. During studies on midecamycin biosynthesis, we found midecamycin A1 and A3 were interconvertible using washed cells of the high-producing strain S. mycarofaciens No. 510-19. Following Suzuki et al's report of interconversion among leucomycin A3, carbomycin A, carbomycin B, and maridomycin II by S. hygroscopicus, we report here the interconversion between midecamycin A1 and A3 by an enzyme system extracted from S. mycarofaciens No. 510-19. Interconversion activity was detected in crude extracts: the conversion of A3 to A1 required NADPH (rather than NADH), and A1 to A3 required NADP (superior to NAD). Reaction products were identical to authentic samples via UV spectrum, bioautography, and NMR data. Highest activities were found in crude extracts from 3-day cultures; the activities were non-dialyzable and heat-labile. Both activities co-fractionated with 30-60% ammonium sulfate and in the same fractions during Sephadex G-100 chromatography, and were not separated by DEAE-Sephadex A-25. Heating the enzyme (from Sephadex G-100) at 50°C for 10 minutes reduced ~75% of both activities. The molecular weight was estimated to be ~40,000 by Sephadex G-100. The optimum pH for reducing the lactone ring of A3 was ~6.0, and for oxidizing A1 was ~7.5. We speculate this oxidoreduction is catalyzed by the same enzyme. The enzyme(s) did not reduce the 18-position aldehyde group of the lactone ring and was undetectable in Streptomyces that do not produce macrolide antibiotics, suggesting involvement in midecamycin biosynthesis.