We have previously shown that an avirulent strain of Salmonella typhimurium, SL3235, blocked in aromatic synthesis, confers high levels of resistance to challenge with virulent Salmonella as early as 3 days postvaccination. In the present studies, it was found that immunization with SL3235 resulted in high levels of natural killer (NK) cell activity in the spleens and peritoneal cavities of C3H/HeJ mice, as measured by cytotoxicity against YAC-1 targets. NK cell activity was at its maximum 2 to 4 days after immunization and was ablated by in vivo or in vitro treatment with anti-asialo GM1. In vivo treatment with anti-asialo GM1 during the first week after immunization with SL3235 depleted NK cell activity and markedly increased mortality in mice challenged with a virulent Salmonella strain. These results are compatible with a role for NK cells as one important component in the resistance against virulent Salmonella infection induced by a live, attenuated vaccine.