Unlike the ⁷⁰-controlled P2 promoter for the osmotically regulated proU operon of Escherichia coli and Salmonella enterica serovar Typhimurium, the ^s-controlled P1 promoter situated further upstream appears not to contribute to expression of the proU structural genes under ordinary growth conditions. For S. enterica proU P1, there is evidence that promoter crypticity is the result of a transcription attenuation phenomenon which is relieved by the deletion of a 22-base C-rich segment in the transcript. In this study, we have sought to identify growth conditions and trans-acting mutations which activate in vivo expression from proU P1. The cryptic S. enterica proU P1 promoter was activated, individually and additively, in a rho mutant (which is defective in the transcription termination factor Rho) as well as by growth at 10°C. The E. coli proU P1 promoter was also cryptic in constructs that carried 1.2 kb of downstream proU sequence, and in these cases activation of in vivo expression was achieved either by a rho mutation during growth at 10°C or by an hns null mutation (affecting the nucleoid protein H-NS) at 30°C. The rho mutation had no effect at either 10 or 30°C on in vivo expression from two other ^s-controlled promoters tested, those for osmY and csiD. In cells lacking the RNA-binding regulator protein Hfq, induction of E. coli proU P1 at 10°C and by hns mutation at 30°C was still observed, although the hfq mutation was associated with a reduction in the absolute levels of P1 expression. Our results suggest that expression from proU P1 is modulated both by nucleoid structure and by Rho-mediated transcription attenuation and that this promoter may be physiologically important for proU operon expression during low-temperature growth.