Several approaches were used to study the transcriptional control region of the melanin-production locus (melC) of Streptomyces antibioticus. Filter-binding in combination with exonuclease III protection localized the 3' boundary of a Streptomyces RNA polymerase-binding site predominantly about 39 nucleotides (nt) upstream from the start codon of melC1, the first open reading frame in the melC locus. Deletion of nt 112-197 upstream from the melC1 start codon reduced melC expression to less than 10%, and deletion of nt 28-107 or 28-120 upstream from melC1 totally inactivated melC. High-resolution nuclease S1 mapping identified the in vivo transcriptional start point (tsp) at 33-34 nt upstream from the start codon of melC1. No sequence resembling the E. coli consensus promoter sequence was found in this region, and site-directed mutagenesis of such a sequence located 101-132 nt upstream from melC1 did not influence melC expression. These studies suggest that transcription of melC is principally from a single tsp and is positively regulated by a mechanism that involves sequences 87-163 nt upstream from the tsp.