An increased incidence of thromboembolic events has been associated with nonionic contrast media. Although it has been demonstrated that nonionic contrast media cause significant platelet activation in vitro, there is no reported evidence of contrast-induced platelet activation in vivo. This study investigated the effect of a nonionic contrast agent, iohexol (Omnipaque^®), versus an ionic contrast agent, diatrizoate meglumine + diatrizoate sodium (MD-76^®), on both in vitro and in vivo platelet activity. Blood from normal subjects (n = 12) was incubated in vitro for 15 to 60 seconds with varying proportions of contrast media or saline solution, and analyzed by flow cytometry for markers of platelet degranulation (P-selectin) and platelet GPIIb-IIIa receptor activation (PAC-1). In vivo activation after Omnipaque was measured in patients undergoing cardiac catheterization in peripheral blood before and after contrast (n = 5), and in coronary sinus blood before and after left coronary injections (n = 8). In vitro data showed that Omnipaque incubated for 60 seconds with whole blood at ratios of 1:1 resulted in a greater percentage of platelets expressing P-selectin (median/ range) than did MD-76 (68%/38% to 90% vs 17%/1% to 53%, respectively, p < 0.01). Similarly at a contrast: blood ratio of 1:3, Omnipaque caused greater P-selectin expression than did MD-76 (49%/19% to 83% vs 3%/0% to 5%, respectively, p < 0.05). There was no significant increase in platelet degranulation observed with concentrations of contrast more dilute than 1:3. In addition, there was significantly less activation seen even at contrast:blood ratios of 1:1 when exposure time was < 60 seconds (p < 0.01). In vivo, there was no increase in peripheral P-selectin (3 ± 1% before vs 2 ± 1% after) and PAC-1 (4 ± 3% before vs 3 ± 3% after), or coronary sinus P-selectin (1 ± 1% before vs 1 ± 1% after) and PAC-1 (3 ± 2% before vs 3 ± 3% after) with nonionic contrast media. In conclusion, Omnipaque causes more significant in vitro platelet activation than MD-76, but only in high concentrations and after prolonged exposure. By the methodology utilized, this study found no evidence of platelet activation in vivo, possibly reflecting the absence of these conditions in the coronary circulation where coronary blood flow rapidly dilutes contrast media.