Objectives: To define clinically relevant reference ('normal') values for cerebrospinal fluid (CSF) protein concentrations in pediatric patients who were evaluated for meningitis by traditional criteria and by enterovirus-polymerase chain reaction (EV-PCR). Design and Patients: A cohort of 906 consecutive pediatric patients to receive CSF analysis at St Louis Children's Hospital, St Louis, Mo, from June 1, 1998, to December 31,1998, was studied for clinical and laboratory data. Age-dependent CSF protein concentrations were then derived from a reference group of 225 patients in whom meningitis and other neurologic diseases were excluded by traditional clinical or laboratory criteria (excluding EV-PCR). Available CSF samples from 132 patients of the reference group were subsequently tested for EV-PCR. Results: In the reference group, the CSF protein concentration was highest and most variable in neonates, with a maximum of approximately 1.0 g/L. Cerebrospinal fluid protein concentration decreased rapidly to a nadir by 6 months and remained low throughout childhood, rarely exceeding 0.3 g/L and, finally, increasing in adolescence toward adult values. Enterovirus-polymerase chain reaction was positive in CSF of 11% of the reference group, with EV-PCR-positive patients having significantly higher CSF protein concentrations than EV-PCR-negative patients aged between 4 months and 14 years. Conclusions: Reference values for CSF protein exhibit a characteristic age dependence in pediatric patients. Continued standard use of adult reference values in the pediatric population is inappropriate. The unexpected finding of a positive EV-PCR in patients not diagnosed with meningitis by traditional criteria further emphasizes the importance of selecting the most clinically relevant reference group for age and other variables when defining normal laboratory values.