A protein serine/threonine kinase, AfsK, and its target protein AfsR globally control physiological and morphological differentiation in the bacterial genus Streptomyces. A protein (KbpA) of 252 amino acids encoded by an open reading frame in a region upstream of afsK in Streptomyces coelicolor A3(2) was identified as an AfsK-interacting protein. The interaction site of AfsK was in the N-terminal portion containing the kinase catalytic domain. KbpA bound a nonphosphorylated form of AfsK and inhibited its autophosphorylation at serine and threonine residues. KbpA in the reaction mixture containing AfsK and AfsR also inhibited the phosphorylation of AfsR by AfsK, presumably because KbpA inhibited the conversion from the inactive, nonphosphorylated form of AfsK to the active, phosphorylated form. kbpA was transcribed throughout growth, and the transcription was enhanced when production of actinorhodin had already started. KbpA thus appeared to play an inhibitory role in a negative feedback system in the AfsK-AfsR regulatory pathway. Consistent with these in vitro observations, kbpA served as a repressor for actinorhodin production in S. coelicolor A3(2); disruption of kbpA greatly enhanced actinorhodin production, and overexpression of kbpA reduced the production.