Streptomyces sioyaensis Lv81 produces siomycin, a thiopeptide antibiotic synthesized on ribosomes. Nothing is known about the ability of this strain to produce nonribosomal peptides, a well represented group of natural actinomycete compounds. Using degenerate primers, we cloned a number of DNA fragments encoding putative adenylation domains (A domains) of nonribisomal peptide synthetases involved in biosynthesis of unknown compounds. Sequencing of amplicons revealed nine different A domains, which were analyzed in more detail. Nonribosomal codes of these domains were determined, but in most cases their substrate specificity failed to be unambiguously predicted. This means that many of these domains can recognize novel amino acids and be used to improve and expand the bioinformatic toolbox applied to predict substrate specificity of A domains. Multiple sequence alingments showed that the cloned A domains are probably involved in different biosynthetic pathways. Five A domains were used in gene inactivation experiments. Inactivation of one of them (in strain 736) resulted in a decrease of the total antibiotic activity as compared to the initial strain. Other A-domain mutants were similar to the initial strain in morphology and siomycin production. The causes of reduced antibiotic activity of strain 736 are discussed.