28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial 28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the 28 -dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a ؊35 element recognized by chlamydial 28 RNA polymerase that resembles the consensus ؊35 sequence. Within the ؊10 element, however, chlamydial 28 RNA polymerase showed a striking preference for a CGA sequence at positions ؊12 to ؊10 rather than the longer consensus ؊10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli 28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the ؊10 promoter element recognized by 28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that 28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11-or 12-nt spacer equally well. Altogether, we found very similar results for 28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred 28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n 11/12 -ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n 11/12 is a spacer of 11 or 12 nt.