Lysostaphin sensitivity was evaluated as a rapid screening test to differentiate Staphylococcus aureus from other species of staphylococci and micrococci. A total of 168 strains of staphylococci, 108 of which were S. aureus, were cultured overnight in brain infusion broth. Gram stains were peformed before and after a 1:10 dilution of the culture was exposed to 2 micrograms of lysostaphin per ml at 37 degrees C for 30 min. A reduction of 90% or greater in the number of organisms seen on comparison of the pre- and posttreatment Gram stains was considered a "positive" test result and was found in 106 of 108 S. aureus strains; 60 of 60 non-S. aureus staphylococci had a negative test result, showing no difference between the pre- and posttreatment Gram stains. Identical results were obtained using commerical blood culture media in place of brain heart infusion broth. Also studied prospectively were 100 blood or broth cultures which the clinical microbiology laboratory identified as containing gram-positive cocci suggestive of staphylococci. All 33 cultures later found to contain S. aureus gave positive test results; 67 of 67 non-S. aureus staphylococci, micrococci, and steptococci were negative.