Listeria monocytogenes is a facultative intracellular pathogen which can escape bactericidal mechanisms and grow within macrophages. The intracellular environment of macrophages is one of the most stressful environments encountered by an invading bacterium during the course of infection. To study the role of the major stress protein, DnaK, of L. monocytogenes in survival under intracellular stress induced by macrophage-phagocytosis as well as under extracellular environmental stresses, we cloned, sequenced, and analyzed the dnaK locus from L. monocytogenes. Then we constructed an insertional mutation in the dnaK gene by homologous recombination and characterized it. Sequencing has revealed that the dnaK locus consists of four open reading frames in the order hrcA-grpE-dnaK-dnaJ. The mutant grows neither at temperatures above 39 degrees C nor under acidic conditions e.g. pH 3.0. Using the macrophage cell line JA-4, the ability of the dnaK mutant to grow intracellularly was examined. Immediately after phagocytosis, the number of viable dnaK mutant bacteria found within macrophages was significantly lower compared to that of intracellular wild type bacteria. However, following a 1-3 h latency period, the mutant multiplied in a similar fashion to the wild type within macrophage cells. A quantitative analysis of intracellular bacteria in macrophage cells by microscope and a binding assay of bacteria to the surface of macrophages by ELISA revealed that the lower number of viable dnaK mutant in macrophages after phagocytosis is due to the low efficiency of phagocytosis resulting from the reduced binding capacity of the dnaK mutant. These results demonstrate that DnaK of L. monocytogenes is essentially required for survival under high temperatures and acidic conditions. Though it does not largely contribute to the survival of L. monocytogenes in macrophage cells, it is essential for efficient phagocytosis. This is the first evidence that DnaK is required for the efficient phagocytosis of a facultative intracellular pathogen with macrophages.