The nucleotide sequence of the tetracycline resistance (tet) gene and its regulatory region, encoded by the plasmid pSTE1 from Staphylococcus hyicus, was determined. The tet gene was inducible by tetracycline and encoded a hydrophobic protein of 458 amino acids. Comparisons between the predicted amino acid sequences of the pSTE1-encoded Tet from S. hyicus and the previously sequenced Tet K variants from Staphylococcus aureus, Tet L variants from Bacillus cereus, Bacillus stearothermophilus, and Bacillus subtilis, Tet M variants from Streptococcus faecalis and Staphylococcus aureus as well as Tet O from Streptococcus mutans were performed. An alignment of Tet amino acid sequences revealed the presence of 30 conserved amino acids among these Tet variants. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated large evolutionary distances between the Tet M and Tet O variants on one hand and the Tet K and Tet L variants on the other hand. The pSTE1-encoded Tet proved to be closely related to the Tet L proteins originally found on small Bacillus plasmids. The observed extensive similarities in the nucleotide sequences of the tet genes and in the deduced Tet amino acid sequences allowed the assignment of the pSTE1-encoded Tet to the Tet proteins of class L.