The recommendation to measure cerebrospinal fluid opening pressure in the extended, rather than the flexed lateral recumbent position to avoid false elevation of the opening pressure has not been formally evaluated in children. This single-center prospective cohort study includes 53 children who had their opening pressure measured in both the flexed and extended lateral recumbent positions prior to removing any cerebrospinal fluid (mean age = 11.7 years; 60% male). The mean opening pressure was higher in the flexed (25.1 +/- 9.2 cm H2O) compared with the extended (24.4 +/- 8.4 cm H2O) position (mean difference = 0.6 +/- 2.2 cm H2O; Z = 2.021, P < .03). Most (92.4%) opening pressure measurements had less than a 5 cm H2O difference between positions. Lumbar puncture performed in the extended, rather than the flexed lateral recumbent position results in a statistically significant decrease in cerebrospinal fluid opening pressure, although the magnitude of the difference is small and of doubtful clinical significance.