When endotoxin low-responder C3H/HeJ mice were immunized with L-form Salmonella typhimurium, the mice were more susceptible to a lethal challenge with S. typhimurium 1 week after immunization (1-week mice) than were the unimmunized controls. One-week immune mice produced overwhelming amounts of tumor necrosis factor-alpha (TNF-alpha) in the blood after infection, while 4-week immune mice produced lesser amounts of this cytokine with a 75% survival rate at 60 days postinfection. Pretreatment with anti-TNF-alpha antibody prevented 1-week immune mice from succumbing to acute illness. Endotoxin-stimulated peritoneal macrophages from 1-week immune mice produced higher amounts of TNF-alpha in vitro than did those from 4-week immune mice and they expressed larger amounts of TNF-alpha mRNA on Northern blot. The capacity of macrophages to produce TNF-alpha in vitro was correlated with the degree of colonization by the L form in the cells. These results suggest that the colonization by L-form S. typhimurium in macrophages alters the susceptibility to S. typhimurium of C3H/HeJ mice and that TNF-alpha might play a major role in this alteration of host resistance.