Measurement of in vivo platelet activation is difficult after phlebotomy and during blood processing for analysis. We used flow cytometry to measure platelet surface expression of P-selectin in the presence and absence of trimethylsphingosine (a platelet activation inhibitor) and compared the results with those from the standard methods of preventing in vitro P-selectin expression. Percent activation was calculated as a ratio of mean sample fluorescence to 100% mean fluorescence after phorbol myristate acetate treatment. Twenty-five micromoles per liter of trimethylsphingosine kept in vitro platelet activation below 5% up to 6 hours after collection and below 10% at 24 hours after collection. Trimethylsphingosine failed to prevent platelet activation caused by centrifugation, storage at 4 degrees C, or stimulation with common agonists. Addition of trimethylsphingosine to whole blood was valuable in preventing in vitro platelet activation. This compound promises to be a useful preservative for diagnostic testing of platelet activation.