Expression of a lac operon in Salmonella typhimurium single cells was monitored using lac mRNA targeting in situ reverse transcription-polymerase chain reaction (RT-PCR). It is demonstrated that suboptimal induction of the lac operon in a culture of S. typhimurium/F′ lac+ cells generates a subpopulation in which transcription of the lac operon occurs and another subpopulation in which transcription of the lac operon is repressed, whereas suboptimal induction of the lac operon in a culture of S. typhimurium/F′ lacY cells generates a population with uniform levels of lac mRNA. The outcome of the single-cell lac mRNA detection assay was compared with the outcome of a single-cell β-galactosidase assay. In cultures grown under different suboptimal lac induction conditions, the fraction of cells in which transcription of the lac operon occurred was concurrent with the fraction of cells showing β-galactosidase activity. Besides supporting the hypothesis that the lactose permease has a role in generating non-genetic heterogeneity in suboptimally induced cultures of Lac+ cells, these results demonstrate the usefulness of in situ RT-PCR for the study of non-genetic population heterogeneities.