In the present study, three Listeria monocytogenes strains and one Listeria innocua strain out of a collection of 241 Listeria isolates from human and food-processing sources were found to display resistance to tetracycline (TC) due to the presence of the tet(M) gene. Through sequence analysis, it was shown that tet(M) genes in two of the isolates belong to sequence homology group (SHG) II, a group comprising chromosomally encoded tet(M) genes previously found in Staphylococcus aureus and in lactobacilli. The tet(M) genes of the two other L. monocytogenes strains were associated with a member of the Tn916-Tn1545 family of conjugative transposons and were closely related to SHG III, which harbours enterococcal tet(M) genes associated with Tn916. One of these transposon-containing strains was able to transfer the tet(M) gene to Enterococcus faecalis recipient strain JH2-2. Collectively, these sequence and conjugation data indicate that the acquisition of tet(M) by Listeria strains may be triggered by successive transfers between other Gram-positive organisms.