We demonstrated two different DNA-dependent RNA polymerase (RNAP) alpha subunits in spores of Streptomyces granaticolor with apparent molecular masses of 40 and 43 kDa. The 43-kDa subunit was also found in vegetative cells. These two proteins are highly similar to each other as well as to other bacterial RNAP alpha subunits. The 40-kDa subunit is shortened from its C-terminus, in the portion of the protein, required for binding of DNA and transcription regulators. The gene for RNAP alpha from S. granaticolor was cloned and sequenced and the corresponding protein was overproduced in Escherichia coli. In vitro experiments using purified RNAP alpha showed that the cell free extract from spores of S. granaticolor exhibits proteolytic activity responsible for the alpha subunit shortening, whereas that from vegetative cells does not. This modification of alpha subunit might point to a novel mechanism of transcriptional control in streptomycetes.