The Caulobacter crescentus flagellar (fla) genes are organized in a regulatory hierarchy in which genes at each level are required for expression of those at the next lower level. To determine the role of this hierarchy in the timing of fla gene expression, we have examined the organization and cell cycle regulation of genes located in the hook gene cluster. As shown here, this cluster is organized into four multicistronic transcription units flaN, flbG, flaO, and flbF that contain fla genes plus a fifth transcription unit II.1 of unknown function. Transcription unit II.1 is regulated independently of the fla gene hierarchy, and it is expressed with a unique pattern of periodicity very late in the cell cycle. The flaN, flbG, and flaO operons are all transcribed periodically, and flaO, which is near the top of the hierarchy and required in trans for the activation of flaN and flbG operons, is expressed earlier in the cell cycle than the other two transcription units. We have shown that delaying flaO transcription by fusing it to the II.1 promoter also delayed the subsequent expression of the flbG operon and the 27- and 25-kDa flagellin genes that are at the bottom of the regulatory hierarchy. Thus, the sequence and timing of fla gene expression in the cell cycle are determined in large measure by the positions of these genes in the regulatory hierarchy. These results also suggest that periodic transcription is a general feature of fla gene expression in C. crescentus.