Endonucleolytic activity was detected in high-salt extracts of Chlamydia trachomatis cell lysates. This nucleolytic activity showed specificity for one strand of a PCR fragment containing the strong chlamydial promoter that regulates synthesis of two short plasmid-specified RNAs. Strand specificity was also observed with supercoiled DNA containing the same region; once again, the same strand was cleaved. The sensitive strand is the nontemplate strand for two short transcripts which initiate at the same nucleotide. Alignment of sequences on either side of the nick sites revealed a consensus which may be required for recognition of the enzymatic activity.