P-selectin is an adhesion molecule found in the alpha granules of platelets. Activation occurs in response to a range of inflammatory and thrombotic agents resulting in rapid up-regulation. Flow cytometry methods have recently been described for the analysis of platelet P-selectin expression in whole blood. While introducing these methods into our laboratory it was noted that expression could be stimulated, in vitro, in a number of ways. This study shows that red cell lysis, the anticoagulant K3 EDTA and the time elapse between blood collection and antibody labelling had statistically significant effects on P-selectin expression. Post-labelling fixation, with CellFIX, caused no significant effect. We conclude that blood for P-selectin analysis should be collected in sodium citrate and that red cell lysis and centrifugation should be avoided. When comparing samples, the time between collection and labelling should be standardized. The relatively high CV for the assay indicates that all samples should be labelled and analysed in duplicate with the mean level reported.