A DNA probe derived from the PBP 2a gene of the methicillin-resistant Staphylococcus aureus COL was compared with phenotypic microbiologic tests for its ability to identify methicillin-resistant and -susceptible staphylococci. Lysates were applied to nitrocellulose with a dot blot apparatus. Isolates tested were both S. aureus and coagulase-negative staphylococci that had been recovered from a variety of geographic and clinical sources. When compared with a spread plate phenotypic test, the DNA probe gave sensitivity, specificity, and predictive values for both positive and negative tests of 100% for 204 S. aureus isolates (103 positive, 101 negative) and 99, 95, 99, and 95%, respectively, for 249 coagulase-negative staphylococci (210 positive, 39 negative). The probe was more sensitive than broth microdilution and more specific than agar dilution in identifying methicillin-resistant and -susceptible coagulase-negative staphylococci; all tests were equally accurate in identifying the methicillin susceptibility of S. aureus. DNA probe analysis for determining the methicillin susceptibility of staphylococci was rapid, easily interpretable, and equally accurate with radioactive and nonradioactive probes, and it gave results equivalent to the most sensitive microbiologic test for all staphylococcus species studied.