Listeria monocytogenes internalin A (InlA) is a surface protein that mediates the attachment of Listeria to, and invasion of, hepatocytes, epithelial, and endothelial cells. In this study, we tested whether InlA could also mediate phagocytosis of L. monocytogenes by the non-listericidal mouse macrophage cell lines J774A.1 and H36.12j. Recombinant InlA (rInlA) was used to derive mouse monoclonal anti-InlA antibodies (mAb) and rabbit anti-InlA antibodies. Fluorescence microscopy demonstrated that these anti-InlA antibodies reacted with wild-type L. monocytogenes, L. ivanovii, and L. innocua+, a mutant transformed with the inlAB operon that expresses surface InlA but failed to react with Bug 8, an InlA/InlB-negative transposon mutant of L. monocytogenes or with noninvasive Listeria sp. Fluorescence microscopy, radiolabeling, and flow cytometry showed that rInlA bound specifically to both macrophage cell lines. Incubation of macrophages and wild-type L. monocytogenes in the presence of rInlA or pretreatment of Listeria with anti-InlA antibodies specifically inhibited, by at least 50%, the phagocytosis of Listeria by both of these cells. By comparison, treatment with these reagents failed to affect the phagocytosis of Streptococcus pyogenes by either macrophage cell line nor did these reagents alter the ability of macrophages to internalize wild-type L. monocytogenes. We found that Bug 8, but not wild-type L. monocytogenes, failed to grow within both of these non-listericidal macrophage cell lines. In contrast to infection by wild-type L. monocytogenes, Bug 8 was rapidly eliminated from the spleens of both C57Bl/6 and DBA/2 mice. Data presented here show that only invasive Listeria sp. have surface InlA and that L. monocytogenes can enter non-listericidal macrophage cell lines by binding of bacterial InlA to the macrophage cell surface.