Lactobacillus acidophilus, L. casei, and L. helveticus are industrially important bacteria for the manufacture of fermented dairy foods. Despite widespread commercial use, there is limited knowledge of basic physiological responses by these bacteria to dairy processing conditions. This study investigated the heat shock (HS) response in L. acidophilus NCFM, L. casei LC301, and L. helveticus LH212. Thermotolerance experiments showed HS improved the ability of log phase L. acidophilus NCFM, L. casei LC301, and L. helveticus LH212 cells to withstand a 20 min high temperature incubation by approximately 27-, 5- and 11-fold, respectively. Two-dimensional polyacrylamide gel electrophoresis showed HS induced synthesis of several proteins in each Lactobacillus species, and Western blots revealed these molecules included homologs to the universally conversed heat shock proteins DnaK, GroEL, ClpB, and GrpE. DnaJ was also detected, but expression of this protein was not stimulated by HS in any of the Lactobacillus species tested.