Seven new sites leading to increased beta-lactam susceptibility were identified and mapped in Staphylococcus aureus by Tn551 or Tn917 mediated insertional inactivation. Their effect was more pronounced in methicillin resistant than in susceptible strains. Except for inserts each in SmaI-K and SmaI-B, all were located on SmaI-A, which covers 25% of the staphylococcal chromosome. The physical position of the femAB operon involved in the expression of methicillin resistance was mapped on SmaI-A. Close to this site we identified a further site with almost as strong an effect on methicillin resistance as femAB. A second cluster of sites affecting methicillin resistance was identified at approximately 30 kb from the femAB operon, showing that multiple unlinked factors may affect beta-lactam resistance. Interestingly, insert Omega2016 located in SmaI-B produced small colonies and was found to inactivate a gene of the electron transport chain, reminiscent of small colony variants observed in recurrent infections.