We report a novel phenotype of methicillin resistance, designated "Eagle-type" resistance, which is characteristic in its resistance to high concentrations of methicillin (64 to 512 µg/ml) and susceptibility to low concentrations of methicillin (2 to 16 µg/ml). The type of resistance was expressed in mutant strains selected with high concentrations (e.g., 128 to 512 µg/ml) of methicillin from the pre-methicillin-resistant Staphylococcus aureus strain N315, whose mecA gene transcription is strongly repressed by the mecI gene-encoded repressor protein MecI. The Eagle-type mutant strains harbored no mutation in the mecI gene or in the operator region of mecA gene to which MecI repressor is supposed to bind. In the representative Eagle-type strain h4, repression of mecA gene transcription and penicillin-binding protein 2' production were found to be released by exposing the cells to a high concentration (128 µg/ml) of methicillin but not to lower concentrations (1 and 8 µg/ml) of methicillin. The strain h4 expressed paradoxical susceptibility (Eagle effect) to the cytokilling activity of methicillin. Experimental deletion of mecI gene from the chromosome of h4 by mecI-specific gene substitution converted its Eagle-type resistance to homogeneously high methicillin resistance. We cloned two novel genes, designated hmrA and hmrB, from genomic library of h4, which conferred Eagle-type resistance to N315 when introduced into the cell in multiple copies. The genes were shown to confer homogeneous methicillin resistance to the heterogeneously methicillin-resistant strain LR5 when they were introduced into on multicopy plasmids. This result strongly indicated that the genetic alteration responsible for the expression of the Eagle phenotype is identical, or equivalent in its effect, to the genetic alteration underlying heterogeneous-to-homogeneous conversion of methicillin resistance in S. aureus.