Aims: To characterize the function of both metK1-sp (sp1190) and metK2-sp (sp1566) in vitro and in vivo, and to study the regulation of doxorubicin production by overexpressing the metK. Methods and Results: We cloned two orfs into pET32a(+) respectively, and the formation of S-Adenosyl-l-methionine was clearly observed in the in vitro enzyme assays as functional MetKs. Reverse transcriptase polymerase chain reaction (PCR) analysis indicated that the transcripts for the metK1-sp were repressed as Streptomyces cells entered the decline phase, whereas that of the metK2-sp was induced, suggesting that these MetK proteins may be important for the growth and the regulation of secondary metabolites during the stationary growth phase, whether considered together or separately. Furthermore, we found that the introduction of high-copy-number plasmids containing the metK1-sp and metK2-sp resulted in 2 center dot 1- and 1 center dot 4-fold greater levels of doxorubicin production than the control transformants containing only the vector, respectively. We also attempted to disrupt the metK-sp and found that doxorubicin production from the metK1-sp-deleted mutant (Streptomyces peucetius/pNN1) was reduced when compared to the parent strain (S. peucetius var. caesius ATCC 27952). Conclusions: The results of this study indicated that two metK are differentially expressed during cell growth, and that the expressions of the two metK genes are differentially regulated under the same conditions. Significance and Impact of the Study: Streptomyces peucetius var. caesius contains two genes, metK1-sp and metK2-sp, which encode functional S-adenosyl-l-methionine synthetase (MetK). The degree of homology (90% identity) found between the two genes shows that metK1-sp and metK2-sp are duplicated genes. Although there is currently no evidence for the relationship of the duplicated metK genes involved in the regulation of doxorubicin production, metK1-sp and metK2-sp may play a role in controlling the stimulation of antibiotic production during secondary metabolism.