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Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS− mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10−6 and 10−7 per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations., Author Summary This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile: adaptation, mutation, and non-mutational preadaptation. Adaptation is easily observed in the laboratory: when a Salmonella culture is grown in the presence of a sublethal concentration of the bile salt sodium deoxycholate (DOC), the minimal inhibitory concentration of DOC increases. Adaptation appears to be associated to multiple changes in gene expression induced by DOC. Mutational bile resistance is also a common phenomenon: plating on agar containing a lethal concentration of bile yields bile-resistant colonies. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. Non-mutational preadaptation, a non-canonical phenomenon a priori, suggests that batch cultures contain rare Salmonella cells whose physiological state permits survival upon encountering bile. The view that non-mutational preadaptation may be a consequence of phenotypic heterogeneity is supported by the observation that Salmonella cultures contain cells that activate stress response genes in the absence of DOC.