Mutants of Salmonella typhimurium lacking DNA adenine methylase are attenuated for virulence in BALB/c mice. LD(50) values of a DNA adenine methylation (Dam)(-) mutant are at least 10(3)- to 10(4)-fold higher than those of the parental strain when administrated by oral or intraperitoneal routes. Dam(-) mutants are unable to proliferate in target organs but persist in low numbers in these locations. Efficient protection to challenge with the virulent parental strain is observed in mice infected with a Dam(-) mutant. Use of the ileal loop assay shows that Dam(-) mutants are less cytotoxic to M cells and fail to invade enterocytes. In the tissue culture model, lack of DNA adenine methylation causes reduced ability to invade nonphagocytic cells. In contrast, no effect is observed either in intracellular proliferation within nonphagocytic cells or in survival within macrophages. The invasion defect of Dam(-) mutants is correlated with a distinct pattern of secreted proteins, which is observed in both PhoP(+) and PhoP(-) backgrounds. Altogether, our observations suggest a multifactorial role of Dam methylation in Salmonella virulence.