The plecomacrolides bafilomycin A1 and B1 (1, 2) and concanamycin A (3), produced by different Streptomyces species, show a unique macrolactone structure with characteristic side chains and exhibit striking biological activities including distinct V-type ATPase inhibition. The biosynthesis of 1 and 2 has been established by feeding experiments with 13C-labelled precursors. Both, bafilomycin (1, 2) and concanamycin (3) feature an "unusual C2 chain extension unit" of as yet unknown origin which was addressed by feeding labelled 2-hydroxy- and 2-methoxymalonyl-derivatives.