Hemorrhage secondary to heparin therapy is one of the most serious risks of extracorporeal membrane oxygenation (ECMO). This study determined those variables which affect the precision of activated clotting time (ACT) measurements used to monitor heparin therapy. Heparin therapy of eight neonatal lambs was monitored during ECMO with two Hemochron 400 machines to determine the effects of intermachine variability, test volume, speed and direction of agitation, and interpersonal variability on ACT values. Test volume (0.25 vs. 0.4 ml) and agitation speed (2 shakes/sec vs. one shake/sec) resulted in significantly different ACT values (p < .05). The mean matched ACT values determined from different agitation directions (vertical vs. horizontal) or the use of a stepper pipette to measure test volume vs. visual estimation of test volume were not significantly different. Variability in ACT test results is significantly increased by variations in test volume or agitation (speed and direction), intermachine variability, and by interpersonnel technique. These variables should be considered when devising protocols for ACT monitoring and management of heparin therapy.