Strains of Staphylococcus aureus resistant to antimicrobial agents are a significant medical problem. Clinical strains commonly contain one or more plasmids, the largest usually carrying multiple resistance genes. Most staphylococcal multiresistance plasmids utilise an evolutionarily common theta-mode replication system that encodes a RepA_N-type initiation protein. Similar initiators are also used by plasmids from other Gram-positive genera. Key features of RepA_N replication systems have been revealed by studies of the S. aureus conjugative multiresistance plasmid pSK41. Control of pSK41 copy number has previously been shown to involve a small antisense transcript, RNAI, which acts primarily as a negative regulator of Rep translation. Such antisense RNA control appears to be a common feature shared by all staphylococcal plasmids using a RepA_N-type replication system. However, the pSK41-like conjugative plasmids differ from non-conjugative staphylococcal multiresistance plasmids by virtue of a small gene, cop, located immediately upstream of, and divergently transcribed from, the rep gene. Cop acts as an additional copy number control element by repressing transcription from the rep promoter. Our studies indicate that expression of cop results from transcripts initiating at the antisense RNA promoter, PrnaI, which escape termination. Thus, PrnaI produces two distinct RNA species, a short abundant countertranscript that represses translation of Rep and Cop mRNA, the product of which represses rep transcription.