Chlamydia is predicted to encode two alternative sigma factors that could provide a mechanism for the regulation of gene expression via alternative forms of RNA polymerase. We have demonstrated that sigma 28, one of these alternative sigma factors, is transcriptionally active. Chlamydial sigma 28 RNA polymerase was reconstituted from recombinant sigma 28 protein and core enzyme that was biochemically isolated from chlamydiae. In an in vitro transcription assay, sigma 28 RNA polymerase transcribed the hctB promoter in a sigma 28-dependent manner. Transcription by sigma 28 RNA polymerase was salt tolerant compared with transcription by sigma 66 RNA polymerase, the major form of chlamydial RNA polymerase. As hctB encodes a histone-like protein that is only expressed late in the developmental cycle, our results suggest that sigma 28 RNA polymerase has a role in the regulation of late gene expression in Chlamydia.