To define the prevalence of antibiotic resistance in Listeria species pathogenic for humans and animals, 1100 isolates (60 from cases of listeriosis and 1040 from food and environment) collected worldwide were screened. Of the 61 tetracycline- and minocycline-resistant strains (37 Listeria monocytogenes), 57 harbored tet(M); 4 non-L. monocytogenes isolates contained tet(S). One Listeria innocua isolate was also resistant to streptomycin and contained the tet(M) and aad6 genes. An L. monocytogenes isolate was trimethoprim-resistant, a characteristic not reported previously in Listeria species, because of the presence of a yet-uncharacterized gene. Three clinical isolates of L. monocytogenes were resistant to low levels of streptomycin. Since the tet(M), tet(S), and aad6 genes are common in enterococci and streptococci, these data suggest transfer from the latter to Listeria species. Uniform susceptibility to tetracycline, minocycline, trimethoprim, and streptomycin cannot be assumed any longer for Listeria species.