The periplasmic nitrate reductase (Nap) from Paracoccus pantotrophus has a role in cellular redox balancing. Previously, transcription from the nap promoter in P. pantotrophus was shown to be responsive to the oxidation state of the carbon substrate. During batch culture, expression was higher during growth on reduced substrates such as butyrate compared to more oxidized substrates such as succinate. In the present study the effect of growth rate on nap expression in succinate-, acetate- and butyrate-limited chemostat cultures was investigated. In all three cases transcription from the nap promoter and Nap enzyme activity showed a strong correlation. At the fastest growth rates tested for the three substrates nap expression and Nap activity were highest when growth occurred on the most reduced substrate (butyrate > acetate > succinate). However, in all three cases a bell-shaped pattern of expression was observed as a function of growth rate, with the highest levels of nap expression and Nap activity being observed at intermediate growth rates. This effect was most pronounced on succinate, where an approximately fivefold variation was observed, and at intermediate dilution rates nap expression and Nap activity were comparable on all three carbon substrates. Analysis of mRNA prepared from the succinate-grown cultures revealed that different transcription initiation start sites for the nap operon were utilized as the growth rate changed. This study establishes a new regulatory feature of nap expression in P. pantotrophus that occurs at the level of transcription in response to growth rate in carbon-limited cultures.