Cerebrospinal fluid (CSF) values in the noninfected neonate are not well-delineated. Studies analyzing these values are inconsistent in the criteria used to define the noninfected population. The purpose of our study was to examine CSF values in neonates in the first 30 days of life in whom infection was more thoroughly excluded than in previous reports. Stringent inclusion criteria defined the noninfected population, and the recently available polymerase chain reaction (PCR) for enteroviruses was used in addition to cultures to help exclude viral disease. Results were also stratified by age in weeks to evaluate for any variability that occurs in CSF values during the first month of life. Neonates were selected from subjects enrolled in two studies on aseptic meningitis. Noninfected infants were identified by the following criteria: (1) atraumatic lumbar puncture (< or = 1000 red blood cells/mm3); (2) no antibiotic therapy before lumbar puncture; (3) sterile blood, CSF and urine bacterial cultures; (4) negative CSF viral culture; and (5) negative CSF PCR for enteroviruses. The mean +/- SD total CSF white blood cell count for 108 noninfected neonates was 7.3 +/- 14/mm3 (95% confidence interval 6.6 to 8.0/mm3) with a median of 4/mm3 and a range of 0 to 130/mm3. There were no significant differences in the mean CSF white blood cell counts among age categories. The application of stringent inclusion criteria and the use of the PCR yielded a population of infants that better represents the noninfected neonate than earlier reports. These values can be used for reference in evaluating the febrile or ill neonate.