Background/Aims:A-factor, a γ-butyrolactone autoregulator, in Streptomyces griseusis involved in the regulation of differentiation and antibiotic production. Here we studied the S. griseusB2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the afsRgene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. Methods:Wild afsRwas cloned to the pHJL401 shuttle vector and was transformed to the S. griseusAFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. Results:Transformation of AFN by a wild afsRrestored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of afsRto the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. Conclusions:AfsR has an effect on the regulation of A-factor production in S. griseus. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.