Lactic acid bacteria have been reported to be useful as a health adjunct and are commonly added to food as the delivery mechanism. The literature contains many conflicting observations for their proposed benefits, and the mechanism of action is undefined. One source of variation is the large number of strains used without proper controls supplemented. Additionally, many of the organisms are not characterized for their acid shock response or the acid-tolerance response, which are known to vary among bacterial species. Our objective was to isolate acid-resistant and bile-resistant variants of Lactobacillus acidophilus and to determine the phenotypic changes. The acid- and bile-tolerant isolates were obtained using natural selection techniques after sequential exposure to hydrochloric acid (pH 3.5 to 7.0) and mixed bile salts. The acid- and bile-tolerant isolates retained their ability to grow at pH 3.5 with 0.3% bile after the selective pressure was removed and reapplied. Isolates varied from their parents for stability in freezing, lactose utilization, protease activity, aminopeptidase activity, plasmid profile, and cell-wall fatty acid profile. These data suggest that the isolated acid- and bile-tolerant isolates possess growth advantages over that of the parents under stress conditions and may be considered as candidates for probiotic strains after further characterization with animal models.