Neisseria gonorrhoeae is capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as a sole source of iron. The gonococcal transferrin receptor is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the order tbpB-tbpA but are separated by an intergenic inverted repeat. First, we demonstrated that tbpB and tbpA are cotranscribed and coregulated from the common upstream promoter that precedes tbpB . Using lacZ transcriptional fusion analysis, we found that tbpB -specific transcripts were more prevalent than tbpA -specific transcripts under iron stressed conditions. We confirmed these results using different varieties of RT-PCR applied to native RNA isolated from wild-type gonococci. The results of all analyses indicated that tbpB -specific transcripts were approximately two-fold more prevalent than tbpA -specific transcripts at steady state. In iron-stressed cultures, the ratio of tbpB - to tbpA -specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand on tbp -specific mRNA levels. We evaluated the function of the intergenic region within the tbpBA operon on the differential expression of the tbp genes using deletion analysis. Removal of the intergenic repeat region resulted in decreased amounts of tbpB -specific transcript. By employing a western blot approach, we found that the absence of the intergenic stem-loop resulted in increased levels of TbpB protein, whereas it had no detectable effect on TbpA protein. Thus, we conclude that the intergenic region functions both as a stabilizing element for tbpB -specific mRNA and as a target site for some unidentified regulatory factor. Primer extension analysis revealed a single transcription start site located 31 by upstream of the translation initiation codon, and confirmed that tbpB is transcribed from an iron-regulated promoter. The sequences upstream of the tbpBA promoter region showed homology to E. coli σ 70 promoter sequences and a potential Fur consensus binding sequence. However, insertion analysis indicated that expression of tbpB involves multiple promoter elements, targeted by Fur and some as-yet-uncharacterized repressor.