We previously reported that deficiency of the lytH gene, whose product is homologous to lytic enzymes, caused the elevation of methicillin resistance in Staphylococcus aureus strain SR17238, a strain of S. aureus with a low level of resistance to methicillin (low-level MRSA) (J. Bacteriol. 179:6294-6301, 1997). In this study, we demonstrated that deficiency of lytH caused the same phenomenon in four other clinical isolates of low-level MRSA, suggesting this deficiency to exist in clinical isolates. We therefore searched the region including lytH in 127 clinical isolates of MRSA by PCR and found one strain, SR17164 (methicillin MIC, 1,600 microg/ml), in which the lytH gene was inactivated by insertion sequence IS1182. lytH::IS1182 was replaced with intact lytH in this strain by integration and excision of the plasmid carrying the lytH region. Recombinants with intact lytH genes showed methicillin MICs of 800 microg/ml, twofold lower than those of the recombinants with lytH::IS1182 and the parent. In addition, S. aureus SR17164, which has a high level of methicillin resistance, had properties similar to those caused by lytH deficiency; that is, the resistance levels of strain SR17164 and lytH-deficient variants from strain SR17238 were not significantly affected by llm inactivation, which greatly lowered resistance levels in most other high-level MRSA strains. These findings suggest that lytH inactivation contributed, to some extent, to the resistance level of S. aureus SR17164. To the best of our knowledge, this strain is the first clinical isolate of MRSA for which the genetic base for high-level resistance has been clarified.