The transport of proline is important for the adaptation of Salmonella typhimurium to osmotic stress because exogenous proline permits the growth of the organism in media of elevated osmotic strength that would otherwise be toxic. Measurements of the rate of [3H]proline transport in S. typhimurium indicated that the organism has two distinct proline permeases, the ProU and the ProP systems, whose activities increase more than fivefold as a consequence of growth in media containing 0.3 M NaCl or 0.47 M sucrose. Transport via a third proline permease, the PutP system, is not affected by the osmotic strength of the medium. We constructed strains that carry fusions of lacZ to proU or proP, genes that are required for the two osmotically stimulated proline transport systems. Assays of beta-galactosidase revealed that the transcription of the proU gene is increased more than 10-fold as a result of exposure of the cells to 0.3 M NaCl, 0.47 M sucrose, or equivalent concentrations of other solutes that are not freely diffusible across the cytoplasmic membrane. Increased transcription cannot be triggered by methanol, ethanol, and glycerol, substances that are freely diffusible across the membrane, suggesting that the signal for increased transcription might be an osmotic gradient across the cytoplasmic membrane. The proP gene does not show transcriptional regulation of sufficient magnitude to account for the stimulation of [3H]proline transport. Thus, the osmotic stimulation of the ProP system might be mediated by some posttranscriptional event.