The control of phosphate-regulated genes in Streptomyces coelicolor is mediated by the two-component system PhoR-PhoP. When coupled to the reporter xylE gene the pstS, phoRP and phoU promoters were shown to be very sensitive to phosphate regulation. The transcription start points of the pstS, the phoRP and the phoU promoters were identified by primer extension. phoRP showed a leaderless transcript. The response-regulator (DNA-binding) PhoP protein was overexpressed and purified in Escherichia coli as a GST-PhoP fused protein. The DNA-binding domain (DBD) of PhoP was also obtained in a similar manner. Both PhoP and its truncated DBD domain were found to bind with high affinity to an upstream region of the pstS and phoRP-phoU promoters close to the -35 sequence of each of these promoters. DNase I protection studies revealed a 29 bp protected stretch in the sense strand of the pstS promoter that includes two 11 bp direct repeat units. Footprinting of the bidirectional phoRP-phoU promoter region showed a 51 bp protected sequence that encompasses four direct repeat units, two of them with high similarity to the protected sequences in the pstS promoter. PHO boxes have been identified by alignment of the six direct repeat units found in those promoter regions. Each direct repeat unit adjusts to the consensus G(G/T)TCAYYYR(G/C)G.