The nucleotide sequence and in vivo transcription start sites for rrnA, one of the two rRNA gene clusters of the eubacterium Caulobacter crescentus, have been determined. Two transcription start sites, a major and minor, for the rRNA gene cluster are located more than 700 nucleotides upstream from the 16 S rRNA gene. Transcription was detected from only the major start site in swarmer cells. But after the swarmer-to-stalked cell transition, transcription was detected from both rRNA start sites and continued throughout the developmental cell cycle when cells were grown in minimal medium. On the other hand, transcription from only the major start site was detected in cells growing in a complex medium. A small open reading frame was found upstream from the rRNA gene transcription start sites and was followed by an inverted repeat sequence. No sequence homology was found between the major rRNA gene transcription start site and the Escherichia coli sigma 70 promoters or the consensus sequence elements reported for C. crescentus fla promoters. However, there were two areas of homology when the major rRNA gene promoter was compared to the nucleotide sequence of the C. crescentus trpFBA promoter. There was a 12 nucleotide sequence centered around the -10 region of both promoters that was closely homologous. In addition, immediately downstream from the transcription start there was a sequence element that was identical in both promoters. These nucleotide sequence elements were not in the temporally expressed fla promoters of C. crescentus.