Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 579 bp gene, pimM, whose deduced product (192 aa) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimM combines an N-terminal PAS domain with a C-terminal helix-turn-helix (HTH) motif of the LuxR type. Gene replacement of pimM from the Streptomyces natalensis chromosome with a mutant version lacking the HTH DNA-binding domain resulted in complete loss of pimaricin production, suggesting that PimM is a positive regulator of pimaricin biosynthesis. Complementation of the DeltapimM mutant with a single copy of pimM integrated into the chromosome restored pimaricin production. The insertion of a single copy of pimM, with its own promoter, into the S. natalensis wild-type strain boosted pimaricin production. Gene expression analyses in S. natalensis wild-type and DeltapimM by reverse transcriptase PCR (RT-PCR) of the pimaricin gene cluster revealed the targets for the PimM regulatory protein. According to these analyses, the genes responsible for initiation and first elongation cycles of polyketide chain extension are among the major targets for regulation. Other pim genes are differentially affected. Interestingly, our results indicate that PimM plays its regulatory role independently of PimR, the first pathway-specific regulator of pimaricin biosynthesis.