BACKGROUND: Methicillin resistance in Staphylococcus aureus is conferred by the mecA-encoded penicillin-binding protein PBP2a. Additional genomic factors are also known to influence resistance levels in strain specific ways, although little is known about their contribution to resistance phenotypes in clinical isolates. Here we searched for novel proteins binding to the mec operator, in an attempt to identify new factor(s) controlling methicillin resistance phenotypes. RESULTS: Analysis of proteins binding to a DNA fragment containing the mec operator region identified a novel, putative helix-turn-helix DNA-binding protein, SA1665. Nonpolar deletion of SA1665, in heterogeneously methicillin resistant S. aureus (MRSA) of different genetic backgrounds, increased methicillin resistance levels in a strain dependent manner. This phenotype could be fully complemented by reintroducing SA1665 in trans. Northern and Western blot analyses, however, revealed that SA1665 had no visible influence on mecA transcription or amounts of PBP2a produced. CONCLUSION: SA1665 is a new chromosomal factor which influences methicillin resistance in MRSA. Although SA1665 bound to the mecA promoter region, it had no apparent influence on mecA transcription or translation, suggesting that this predicted DNA-binding protein modulates resistance indirectly, most likely through the control of other genomic factors which contribute to resistance.