Methicillin-resistant Macrococcus caseolyticus strains from bovine and canine origins were found to carry a novel mecD gene conferring resistance to all classes of β-lactams including anti-MRSA cephalosporins. Association of β-lactam resistance with mecD was demonstrated by gene expression in S. aureus and deletion of the mecD-containing island in M. caseolyticus. The mecD gene was located either on an 18,134-bp M. caseolyticus resistance island (McRImecD-1) or a 16,188-bp McRImecD-2. Both islands were integrated at the 3' end of the rpsI gene, carried the mecD operon (mecD-mecR1m-mecIm), and genes for an integrase of the tyrosine recombinase family and a putative virulence-associated protein (virE). Apart from the mecD operon, that shared 66% overall nucleotide identity with the mecB operon, McRImecD islands were unrelated to any mecB-carrying elements or staphylococcal cassette chromosome mec. Only McRImecD-1 that is delimitated at both ends by direct repeats was capable of circular excision. The recombined excision pattern suggests site-specific activity of the integrase and allowed identification of a putative core attachment site. Detection of rpsI-associated integrases in Bacillus and S. aureus reveals a potential for broad-host range dissemination of the novel methicillin resistance gene mecD.