The gene (aphA-5c) encoding for aminoglycoside-O-phosphoryltransferase (APH(3')-Vc) from Micromonospora chalcea 69-683 was cloned by expression in Streptomyces lividans; it showed strong nucleic and amino acid similarities with previously described streptomycetes aph genes. S1 mapping of the. 5' region indicated that, as with several streptomycete genes, transcription apparently initiates at the translation start codon, with no extragenic ribosome binding site. Comparison of the flanking sequences of the actinomycete aph genes indicates considerable divergence, which is not consistent with the notion that clustered biosynthetic genes for structurally related (or identical) antibiotics are disseminated in their entirety between microbial species.