Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvg regulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed at 10 min following an inducing signal, while ptx is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those of fha and ptx. We have identified cis-acting sequences necessary for expression of prn in B. pertussis by using prn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of prn to activate transcription from the promoter. Our genetic data indicate that the region critical for prn activation extends upstream to position -84. However, these data do not support the location of the prn transcription start site as previously published. We used a number of methods, including prn-lac fusions, reverse transcriptase PCR, and 5' rapid amplification of cDNA ends, to localize and identify the bvg-dependent 5' end of the prn transcript to the cytosine at -125 with respect to the published start site.