Penicillinase testing is required for Staphylococcus aureus isolates with a penicillin MIC of </=0.12 mg/L. This study compared five phenotypic assays with a PCR for blaZ when testing 197 S. aureus isolates. The starch-iodine plate method and nitrocefin tests had low sensitivities of 42.9% and 35.7%, respectively. The cloverleaf assay and the penicillin zone-edge determination method had sensitivities of 67.8% and 71.4%, respectively, and these methods might be appropriate in many circumstances, but were not as sensitive as blaZ PCR.