The major flagellar proteins, including the flagellins and the hook protein, are synthesized periodically in the Caulobacter crescentus cell cycle at the time of flagellum assembly. Although fla genes are regulated at the transcriptional level [Ohta, N., Chen, L.-S., Swanson, E. & Newton, A. (1985) J. Mol. Biol. 186, 107-115], the 5' regulatory regions of C. crescentus genes have not been identified. We describe here the results of nuclease S1 protection assays that map the 5' ends of mRNAs synthesized in vivo from transcription units II (hook operon) and II.1 of the hook gene cluster and locate the corresponding promoter regions PII and PII.1. The two promoters are regulated with different periodicities in the cell cycle and have different genetic requirements for expression. The failure to detect transcripts from either PI or PII in Escherichia coli suggests that developmentally regulated promoters of C. crescentus have different recognition sequences from those of E. coli. There is little nucleotide sequence homology between PII and PII.1. There are, however, three regions of homology between PII and the nucleotide sequence 5' to the 29-kDa-flagellin-related gene, and two of these are in regions of dyad symmetry. We discuss the possibility that DNA-protein interactions at homologous nucleotide sequences like those identified in PII are part of a regulatory gene cascade that participates in timing fla gene expression in the C. crescentus cell cycle.