We investigated the relationship between platelet function analyzer (PFA-100) closure times (CT) and bleeding time (BT), platelet aggregation (PA) induced by ADP, arachidonic acid, and collagen, blood cell counts, and von Willebrand factor (VWF) in 120 well-characterised healthy individuals. Pre-analytical and analytical conditions were standardised comprehensively. In a substantial number of cases the differences between duplicate measurements exceeded 15%. The reference range (5th and 95th percentiles) for CT with the collagen/epinephrine (CEPI) and the collagen/ADP (CADP) cartridge was 93-223 s and 64-117 s respectively. Re-examination of 11 individuals with CEPI-CT above the 95th percentile revealed considerable batch-to-batch variation of CEPI-CT. Males had significantly longer CADP than females (P = 0.002). CEPI and CADP-CT measured pm were significantly longer than corresponding values determined am (P = 0.003 and P < 0.0001 respectively). Blood group O was associated with greater CEPI and CADP-CT and lower VWF levels compared with non-O blood groups (P = 0.008, P = 0.0003 and P < 0.0001 respectively). Linear regression analysis revealed association between CEPI-CT, CADP-CT and VWF (P < 0.0001), but no relationship was found between CT and BT or between CT and PA. We conclude that VWF plasma levels modulate PFA-100 CT to a greater extent than platelet function. Establishment of reliable reference ranges and careful standardisation of pre-analytical and analytical conditions is a prerequisite for obtaining reliable PFA-100 results. Duplicate measurements are necessary.