A total of 16 epidemiologically unrelated macrolide-resistant staphylococcal isolates of various animal origins were investigated for the molecular basis of macrolide resistance with respect to previous contact of their host animals with macrolides and lincosamides. All isolates carried ermC-encoding plasmids of 2.3-4.0 kbp. The eight plasmids of staphylococci from animals which had not received macrolides or lincosamides showed inducible ermC gene expression and did not exhibit alterations in the ermC regulatory region. The remaining eight plasmids expressed the ermC gene constitutively. Six of these plasmids were from staphylococci from animals which had received tylosin or spiramycin as feed additives or lincomycin for therapeutic purposes. All constitutively expressed ermC genes revealed either sequence deletions or sequence duplications in their ermC regulatory region, as detected by a PCR assay and by sequence analysis. These sequence deletions and duplications found in naturally occurring plasmids corresponded closely to the mutations seen in the ermC-encoding plasmids after growth of an inducibly resistant strain in the presence of non-inducing macrolides or lincosamides under in vitro conditions.