Thrombin and poly-l-lysine alter the incorporation of acetate, glycerol, and fatty acids into the lipids of washed human platelets. Both aggregating agents decrease the incorporation of acetate into all lipid classes other than free fatty acids. Similarly, glycerol incorporation into complex lipids is impaired by both thrombin and polylysine. Thrombin caused marked depression of the incorporation of palmitic acid into both lecithin and triglycerides. By contrast it enhanced the incorporation of oleic acid into lecithin, but not into triglycerides. The data suggest that the process of primary platelet aggregation is associated with a defect in the assembly of complex lipids.