In blood both tissue-type plasminogen activator (t-PA) and its fast acting inhibitor PAI-1 are present. Upon blood collection and plasma preparation the reaction between these components is still possible, leading to an erroneously low determination of t-PA activity. In this study the effect of handling speed, temperature and anticoagulant on the measured t-PA activity is determined. It was found that handling speed, temperature and type of anticoagulant and PAI-1 level have influence on the t-PA activity measured. Rapid acidification appears the method of choice: Stabilyte vacutainers are the most suitable way for blood collection for t-PA activity measurement.