This work describes a novel plasmid pSPI 87 (5550 bp) carrying the small multidrug resistance determinant smr encoding resistance to quaternary ammonium compounds (QACs). pSP 187 was identified in a Staphylococcus pasteuri isolate recovered from bulk milk in a dairy cattle herd in Norway. Sequence analysis revealed 6 putative ORFs in addition to the smr gene within a cassette with identical genetic organization to that found in the pSK41-like Staphylococcus aureus plasmid pTZ22. A protein homology search suggested the gene product of ORF7 to be a putative replication initiation protein, while ORF2 was predicted to encode a protein homologous to members of FtsK/SpoIIIE cell division-DNA segregation protein families. Sequence similarities to some initiator proteins of rolling circle replicons (RCR) indicated that pSP187 uses a RCR mode of replication, supported by the detection of intermediate ssDNA using SI nuclease treatment and hybridization analysis. Interestingly, a 30-bp sequence found upstream from ORF7 showed high similarity to other dyad symmetry motifs proposed as putative double-strand origins of replication in the plasmids pG13 (Bacillus thuringiensis), pSTK1 (Bacillus stearothermophilus), and pERI-2 (Streptococcus thermophilus). In conclusion: The novel smr-containing plasmid pSP 187 is the first member of RCR group VI to be identified in a Staphylococcus sp. (c) 2006 Elsevier Inc. All rights reserved.