The emergence of antibiotic resistance among important foodborne pathogens like Escherichia coli O157:H7 has become an important issue with regard to food safety. In contrast to the case for Salmonella, antibiotic resistance has been slow in its development in E. coli O157:H7 despite the presence of mobile antibiotic resistance genes in other E. coli organisms that inhabit the same animal host. We set out to determine if rumen fluid influences the transfer of plasmid-mediated, antibiotic resistance to E. coli O157:H7. A commensal E. coli strain from a dairy cow was transformed with conjugative R plasmids and served as the donor in matings with naladixic acid-resistant E. coli O157:H7. R plasmids were transferred from the donor E. coli strain to E. coli O157:H7 in both Luria-Bertani (LB) broth and rumen fluid. R plasmids were transferred at a higher frequency to E. coli O157:H7 during 6 h of incubation in rumen fluid at rates comparable to those in LB broth, indicating that conditions in rumen fluid favor the transfer of the plasmids to E. coli O157. This finding suggests that the cow's rumen is a favorable environment for the genetic exchange of plasmids between microflora and resident E. coli O157:H7 in the bovine host.