During a study of florfenicol-resistant porcine staphylococci from Denmark, the genes cfr and fexA were detected in the chromosomal DNA or on plasmids of Staphylococcus hyicus, Staphylococcus warneri, and Staphylococcus simulans. A novel variant of the phenicol resistance transposon Tn558 was detected on the ca. 43-kb plasmid pSCFS6 in S. warneri and S. simulans isolates. Sequence analysis of a 22,010-bp segment revealed that the new Tn558 variant harbored an additional resistance gene region integrated into the tnpC reading frame. This resistance gene region consisted of the clindamycin exporter gene lsa(B) and the gene cfr for combined resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics bracketed by IS21-558 insertion sequences orientated in the same direction. A 6-bp target site duplication was detected at the integration site within tnpC. Transpositionally active forms of the IS21-558 element, known as minicircles, were detected by PCR and suggest that this insertion sequence is involved in the mobility of the multiresistance gene cfr. Based on the knowledge of the transposition pathways of IS21-like insertion sequences and the sequence features detected, the resistance gene region of plasmid pSCFS6 is believed to have developed via IS21-558-mediated cointegrate formation. The data obtained in this study identified the multiresistance gene cfr not only in three novel host species but also in a novel genetic context whose further analysis suggested that insertion sequences of the type IS21-558 are likely to be involved in the dissemination of cfr.