The methicillin resistance determinant (mec) in Staphylococcus aureus resides on additional DNA not present in isogenic sensitive cells. However, besides mec, other chromosomally determined factors are essential for expression of methicillin resistance. We cloned and characterized a chromosomally determined gene which encodes a factor essential for the expression of methicillin resistance (femA) in S. aureus. femA mapped in chromosomal segment number 18, genetically very distant from the methicillin resistance determinant (mec). The product of femA was a protein of an apparent size of 48 kDa. FemA restored methicillin resistance in S. aureus that had become sensitive to methicillin by insertion of omega 2003 (femA::Tn551). Although FemA was needed for cell growth in the presence of beta-lactam antibiotics, it had no influence on the synthesis of the low affinity, additional penicillin-binding protein (PBP2') encoded by mec and known to be essential for cell wall synthesis in the presence of inhibitory concentrations of methicillin. Nucleotide sequence analysis, Northern RNA blotting and S1 nuclease RNA mapping suggested that femA was transcribed on a polycistronic mRNA. This mRNA contained the coding region (ORF419) producing a protein of 47 kDa. The nucleotide and amino acid sequence of FemA showed homologies with ORF419, suggesting that these genes arose by gene duplication. In addition we present evidence for a second chromosomal factor, femB, involved in expression of methicillin resistance which maps close to femA.