Previously, we have reported that dietary fatty acids can modify the thromboxane A2-dependent activation of rat platelets. Here, we present evidence that this dietary effect is part of a more general effect on platelet signal transduction, putatively involving structural changes in the platelet membranes. Four experiments were performed, where Wistar rats were fed with a high-fat diet enriched in either saturated, n-6 polyunsaturated or n-3 polyunsaturated fatty acids, or with a low-fat diet enriched in n-6 polyunsaturated fatty acids. The type of diet hardly influenced mean number of double bonds in the major platelet phospholipids. Platelet membranes from the rats fed with the saturated-fat diet had phospholipids with relatively high levels of arachidonate, but were low in cholesterol/phospholipid ratio. When compared to this diet group, platelets from other groups had an arachidonate content that was 21 to 47% lower and a cholesterol/phospholipid ratio 3 to 5% higher. The saturated-fat diet resulted in platelets that, in general, were less responsive to agonists than the platelets from other groups: with thrombin, collagen and thromboxane A2 analogue U46619, both early (shape change and phospholipase C-dependent rise in [Ca2+]i) and late (exocytosis and aggregation) responses were relatively low. However, platelet activation evoked by ADP was not influenced by diet type. When the cholesterol content of rat platelets was modified in vitro, it appeared that the early and late responses to thrombin and U46619 increased with the cholesterol/phospholipid ratio. Taken together, these results suggest that in rat platelets (i) the membrane cholesterol/phospholipid ratio can be modulated by a diet rich in saturated fatty acids, explaining, at least in part, the dietary effect on phospholipase C-mediated platelet activation, and (ii) relatively small changes in cholesterol content can have a more profound effect on platelet activation than substantial changes in arachidonate level.