Resistance to activated protein C (APC) has been associated with thrombotic disease in adults. In over 90% of patients, the basis for the APC resistance is a mutation in the coagulation factor V gene that renders the protein less readily inactivated by APC (FV Leiden). We retrospectively analyzed 32 patients who had a thromboembolic event between 1993-1995, for whom whole blood and clinical records were available. The age of the child, location of the thrombus, underlying diseases, and the results of the hypercoagulable evaluation were obtained from hospital and clinic records. Screening for the factor V Leiden mutation was performed on DNA extracted from stored whole blood by amplification of a 267 bp fragment of the factor V gene at the site of the Leiden mutation. The fragment was digested with a restriction endonuclease (Mnl I), allowing for identification of normal, heterozygous or homozygous genotypes. There were 20 children and 12 neonates in the study. Nineteen of the patients had an arterial thromboembolic event and 13 had a venous thrombotic event. Eight of the 32 patients studied were heterozygous for the FV Leiden mutation, none were homozygous. Of 18 CNS events, 16 were arterial (CVA). Four of 16 patients who had a CVA were positive for the FV Leiden mutation, including 2 of 7 (29%) neonates. Of those patients who had a DVT, 4 of 13 were FV Leiden positive. None of the 32 patients were protein C deficient, 10% had protein S deficiency, 3% were antithrombin III deficient and 15% had anti-phospholipid antibodies. The prevalence of the factor V Leiden allele in the general population is 2 to 5% and in adults who have had a DVT about 25%. In this study the prevalence of the factor V Leiden mutation in those subjects who had a thromboembolic event was 25%; 17% for neonates vs. 30% for children. In contrast to adult studies, the prevalence of the FV Leiden mutation in those who had a CVA was 25%, with a comparable prevalence of 31% in those who had a DVT. These data suggest that all neonates and children who have had an arterial or venous thrombotic event should be tested for the factor V Leiden mutation.