In the recent decades, coagulase-negative staphylococci (CNS) have emerged as important nosocomial pathogens with the ability to develop resistance to antibiotics and disinfectants. Multidrug-resistant CNS are currently a common finding in hospital settings and hospitalized patients. Little is known about the occurrence and persistence of multidrug-resistant CNS in animal clinics. A survey of the environmental bacterial flora in a small animal clinic showed a predominance of bacterial species commonly isolated from skin and feces of warm-blooded animals and the environment. At samplings separated by 3 years, multidrug-resistant Staphylococcus haemolyticus was isolated from the floor of four separate animal cages and from a cat's postoperative wound infection. Pulsed-field electrophoresis, multilocus enzyme typing, susceptibility testing, and genotypic characterization suggested that the multidrug-resistant S. haemolyticus isolates belonged to an epidemiological clone. All isolates harbored a chromosomal copy of the mecA gene, a 45-kb plasmid harboring the blaZ and qacA/B determinants, and all except one isolate carried multiple plasmids in the size range 1-22 kb, of which those <5 kb encoded resistance to tetracycline (tetK), macrolides (ermB), and chloramphenicol (cat). One isolate carried a chromosomal copy of the bifunctional gene aacA-aphD conferring resistance to gentamicin. The isolate that was deficient of small plasmids had reverted to a macrolide and chloramphenicol-susceptible phenotype, but had retained its tetracycline resistance due to IS257-mediated integration of the tetK plasmid into the mec region of the chromosome. This finding illustrates bacterial intracellular mobility of resistance genes in natural environments, and highlights the role of insertion sequences in the evolution of multidrug resistance islands on the bacterial chromosome.