We investigated 42 plasmas prepared at different centrifugation speeds with three activated protein C (APC) resistance methods. The APC ratio for fresh platelet-poor plasma declined significantly after freezing and thawing. This effect was more evident with the original method (average reduction 11.3%) than with either the home-made (3.8%) or the modified method (3.2%). No significant decrease in the APC ratio was observed after freezing and thawing of platelet-free plasmas from the same patients. When frozen platelet-poor plasma was centrifuged at high speed after thawing and before testing, there was no significant decrease in the APC ratio, in comparison with fresh platelet-poor plasma using the home-made and modified methods.