The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes. To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII-ORF4, the pathway-specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium-dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII-ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway-specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models.