Murine peritoneal macrophages bind to Salmonella typhimurium in vitro in the absence of exogenous opsonins. We have identified an outer membrane protein of S. typhimurium that mediates this adhesion. Biotin-labeled macrophages were used to probe electroblotted envelope proteins of S. typhimurium that had been previously resolved by polyacrylamide electrophoresis under denaturing and reducing conditions. Macrophages bound to an outer membrane protein with an apparent molecular mass of 44 kDa. The protein was purified to homogeneity and free of detectable lipopolysaccharide. Limited microsequencing of this protein resulted in a 15-amino acid query sequence of A-E-V-Y-N-K-D-G-N-K-L-D-L-Y-G, which shares complete identity with a 15-mer of both the OmpD of S. typhimurium SH 7454 and the OmpC polypeptide of Escherichia coli K-12. Picomolar concentrations of this purified protein significantly inhibited the subsequent adherence of 35S-labeled S. typhimurium to macrophages in monolayers. We propose that this 44-kDa protein is involved in the recognition of S. typhimurium by macrophage during the initial stages of infection.