One hundred reference strains and 1,240 clinical isolates representing 26 species of the family Micrococcaceae were used to evaluate the potential of tests for synergistic hemolysis, adherence to glass, pyroglutamyl-II-naphthylamide hydrolysis, and susceptibility to a set of five antimicrobial agents for differentiating these species and strains within the species. Sixty-eight percent of the clinical isolates exhibited synergistic hemolysis; 69% of the clinical staphylococci but none of the micrococci or stomatococci were adherence positive, and 92% of the strong positive adherence reactions were produced by strains of Staphylococcus epidermidis. Strains from 15 of the species were pyroglutamyl-4-naphthylamide positive, but this test separated Staphylococcus xylosus from other novobiocin-resistant staphylococci and Staphylococcus intermedius from other coagulase-positive species. A polymyxin B disk helped differentiate S. epidermidis from most other coagulase-negative staphylococci, and a bacitracin disk (10 U) helped differentiate Staphylococcus haemolyticus from most other novobiocin-susceptible staphylococci. All strains that were susceptible to furazolidone and resistant to Taxo A disks (bacitracin, 0.04 U; BBL Microbiology Systems, Cockeysville, Md.) were staphylococci. We observed a 91% correlation between species identification obtained with the Staph-Ident system (Analytab Products, Plainview, N.Y.) and conventional methods; but the micrococci and stomatococci were incorrectly identified as staphylococci with Staph-Ident, and several isolates of S. epidermidis were misidentified as Staphylococcus hominis because they were alkaline phosphatase negative. Both these problems can be prevented by adding the simple tests we describe to those already recommended when the Staph-Ident system is used to identify isolates of gram-positive, catalase-positive cocci.