Cerebrospinal fluid parameters are of great importance in diagnosing meningitis, but normal values for preterm neonates are based on small, single-center studies. We sought to determine current values for preterm neonate cerebrospinal fluid parameters and assess the association of cerebrospinal fluid parameters with culture proven meningitis. We performed a cohort study of the first lumbar puncture from 4632 neonates < 34 weeks' gestation performed in the years 1997 to 2004 at 150 neonatal intensive care units managed by the Pediatrix Medical Group. We identified 95 cases of meningitis from the 4632 lumbar punctures. The area under the receiver operating characteristic curves for white blood cell count, glucose, and protein were 0.80, 0.63, and 0.72, respectively, for prediction of culture-proven meningitis. Cerebrospinal fluid parameters used to diagnose meningitis in the absence of dependable cerebrospinal fluid cultures are unreliable. Caution should be employed when interpreting cerebrospinal fluid parameters in the premature neonate.