To determine the penetration of the antistaphylococcal antibiotics, nafcillin, methicillin, and cefazolin, into brain tissue, we gave to each of 27 patients undergoing craniotomy and brain biopsy one of the antibiotics in a 2-g intravenous infusion just before operation. At the time of brain tissue removal (30 to 225 minutes after the start of the infusion). a serum specimen was obtained, and tissue and serum were assayed for antibiotic concentration. Eleven of 13 brain specimens contained detectable nafcillin concentrations between 0.36 and 11 μg/g of tissue (mean. 2.7 μg/g for all 13 specimens). Fourteen of 18 brain tissue specimens contained detectable methicillin concentrations between 0.56 and 5.0 μg/g of tissue (mean, 2.0 μg/g for all 18 specimens). Ten of 11 brain tissue specimens contained detectable cefazolin concentrations between 2.0 and 40 μg/g of tissue (mean, 10.6 μg/g for all 11 specimens). Each antibiotic penetrated “abnormal” brain tissue better than “relatively normal” brain tissue. Because nafcillin is more active against Staphylococcus aureus, we conclude that nafcillin is preferable to methicillin for the therapy of central nervous system staphylococcal infections. Cefazolin achieves higher brain tissue concentrations than the penicillins, but has not been clinically evaluated for the therapy of central nervous system infections.