Objective To evaluate the efficacy of local instillation of tissue plasminogen activator (tPA) for restoring function to occluded central venous catheters (CVCs) in the neonatal population. Study design This was a retrospective review of patients admitted to the neonatal intensive care unit during September 2000 to April 2006 who received instillation of tPA for occluded CVCs. Results Among 18 infants who received tPA for occluded CVCs, gestational age at birth was 32.5 weeks, birth weight was 1550 g, and gestational age at tPA administration was 39 weeks, with 4 neonates ≤32 weeks' gestational age at tPA administration; age at tPA use was 39 days (medians reported). Ten of 18 (55%) of CVC occlusions were successfully opened after using tPA. No bleeding complications of tPA were noted, including increased or new-onset intraventricular hemorrhage, overt bleeding, or changes in INR or partial thromboplastin time attributed to tPA use. Three patients (16.5%) had bacteremia within 7 days of tPA administration, and no catheter ruptures occurred. Conclusions Local instillation of tPA was successful in restoring function to occluded CVCs in a significant proportion of neonates, although success rates were lower than that reported in populations of older age. Despite the vulnerability of neonates to the potential complications of tPA, no major complications were detected even among premature infants.