OBJECTIVE Staphylococcal species are the most common cause of nosocomial infections in the neonate. Because of staphylococcal resistance patterns, vancomycin has become the drug of choice for treatment. Although the blood stream is the usual site of infection, premature infants are at increased risk for the development of meningitis. The aim of this study was to determine vancomycin cerebrospinal fluid (CSF) concentration and penetration following intravenous (IV) administration in critically ill premature infants. METHODS A multiple-dose, open-label, case series was performed at a level III neonatal intensive care unit in a university teaching hospital. Three critically ill premature infants, 26 to 31 weeks of gestation requiring a course of IV vancomycin for suspected or proved sepsis were studied. Vancomycin was administered intravenously at 20 mg/kg, every 18 to 24 hours over 60 minutes. Serum and CSF vancomycin concentrations were obtained and pharmacokinetic analysis and CSF penetration was calculated. RESULTS Serum vancomycin pharmacokinetics were consistent with those previously reported. CSF vancomycin concentrations ranged from 2.2 to 5.6 micrograms/ml and the calculated vancomycin CSF penetration ranged from 26% to 68%. CONCLUSIONS CSF penetration of vancomycin after IV administration was much higher than that reported in older infants and children. This higher penetration may improve clinical outcomes in neonates with central nervous system infections. These data should be encouraging to clinicians who choose to use IV vancomycin for neonatal meningitis.