In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. PfliQ is activated earlier than PccrM. Phosphorylated CtrA (CtrA approximately P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in PccrM significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrM expression. Our data indicate that differences in the affinity of CtrA approximately P for PfliQ and PccrM regulate, in part, the temporal expression of these genes. However, the timing of fliQ transcription but not of ccrM transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA approximately P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA approximately P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle.