Objective: Studies in adults indicate that ampicillin, in a dose-dependent manner, impairs platelet function and moderately prolongs the bleeding time (generally by 60 to 90 s). Unlike aspirin, the inhibition induced by ampicillin involves both reversible and irreversible mechanisms and is not observed immediately after initial dosing (generally requiring approximately 24 h). Ampicillin is administered commonly to neonatal intensive care unit (NICU) patients, but its effect on bleeding time in this population has not been reported earlier.Study Design: We performed neonatal template bleeding times and platelet function analyzer (PFA)-100 tests on 15 NICU patients before and at various intervals after intravenous ampicillin dosing.Result: Neonates were only studied if no beta-lactam antibiotics were administered to their mother during labor, and if they had ampicillin ordered by the clinician at a dose of 50 to 100 mg kg -1 every 12 h. Subjects ranged from 33 to 41 weeks gestation and weighed 1760 to 3835 g. Bleeding times before the first ampicillin dose (n ¼ 15) averaged 134 s (95% confidence interval (CI), 120 to 148 s) and PFA-100 times averaged 123 s (95% CI, 96 to 149 s). After the first dose of ampicillin (n ¼ 5), bleeding times and PFA-100 times did not increase, but after the third (n ¼ 5) and fourth doses (n ¼ 4) bleeding times lengthened by an average of 60 s (95% CI, 37 to 83 s, P<0.001) and PFA-100 times lengthened by an average of 20 s (95% CI, À20 to 60 s, P ¼ 0.15).Conclusion: Ampicillin administered intravenously to NICU patients prolongs the bleeding time, with a magnitude-of-effect and time-to-effect similar to that shown earlier in adults.