We reviewed the isolation of fungi from cerebrospinal fluid (CSF) cultures at Texas Children's Hospital during the past 6 years to evaluate the significance of a positive culture and to identify potential risk factors. Thirty-seven fungal isolates were recovered from 23 patients representing 2% of all 1498 positive CSF cultures for the study period. Candida species accounted for 94.5% of all fungal isolates. Nine of the 23 patients were newborns and 8 of these were very low birth weight premature neonates. C. albicans was recovered from the CSF of all newborns. Eleven patients were children 4 months to 14 years old. Three patients had positive cultures of CSF obtained on postmortem examination. Leading potential risk factors for positive CSF cultures from neonates included antimicrobial therapy, prematurity, very low birth weight, umbilical catheterization, total parenteral nutrition, intubation and respiratory distress syndrome. For children beyond the newborn period, potential risk factors were antimicrobial therapy for concurrent bacterial infection, chronic systemic or central nervous system disease and central venous catheterization. Disseminated fungal infection was documented in 40% of all patients with positive CSF cultures. Fungi recovered from 7 (35%) of 20 live patients were considered contaminants. We conclude that true fungal meningitis in children is accompanied by multiple positive cultures from CSF or CSF and a second site. A single positive CSF culture far fungi should be considered significant when both CSF findings compatible with meningitis and associated risk factors are present. The isolation of fungi from a single CSF culture can be considered insignificant when CSF findings are within normal limits despite the presence of potential risk factors or vice versa. The recovery of unlikely fungal pathogens from CSF should be evaluated on an individual basis.