Technical Abstract: Escherichia coli O157:H7 and Salmonella are widely recognized as important agents of foodborne disease with worldwide distribution. The use of ionophores in feeding growing ruminants is widespread in the United States and has attracted recent interest due to the apparent temporal relationship between initial ionophore use and the increase in human E. coli O157:H7 cases. Four Salmonella serotypes (Derby, Dublin, Enteriditis, and Typhimurium and two strains of E. coli O157:H7 (ATCC 43895 and FDIU 6058) were incubated in the presence of varying concentrations of ionophores (monensin, lasalocid, laidlomycin propionate, and bambermycin) in pure and mixed ruminal fluid cultures. Bacterial growth rates in pure culture were not affected (P > .10) by ionophores at concentrations as high as 10-times the approximate rumen ionophore concentration under normal feeding regimes. Likewise, ionophores had no effect (P > .10) on Salmonella or E. coli CFU plated from 24 h ruminal fluid incubations. Ionophore treatment decreased (P < .01) the acetate: propionate ratio in ruminal fluid cultures as expected. Ionophores had no effect on the foodborne pathogens Salmonella and E. coli O157:H7 in vitro. Results suggest that ionophore feeding would have little or no impact on Salmonella or E. coli populations in the ruminant.