This study investigated the role of platelet protein synthesis in platelet aggregation. Cycloheximide (Cx) and chloramphenicol (Cm) were used as inhibitors of cytoplasmic (80S ribosome directed) and mitochondrial protein synthesis respectively. The effect of these agents on human platelet aggregation and L-[U-14C]leucine incorporation into platelet protein was investigated. Cm exhibited dose-dependent inhibition of collagen, thrombin and secondary ADP aggregatory responses, but had no effect on arachidonate or primary ADP responses over a similar concentration range (3.1 X 10(-3), 3.1 X 10(-4) and 3.1 X 10(-5) M). Cm also inhibited platelet secretion associated with collagen and secondary ADP responses. Furthermore, Cm exhibited a similar dose-dependent inhibition of L-[U-14C]leucine incorporation into platelet protein reaching 80% inhibition of incorporation at 3 X 10(-3) M. At similar concentrations (3.5 X 10(-3), 3.5 X 10(-4) and 3.5 X 10(-5) M) Cx failed to show inhibition of human platelet aggregation by all agonists used with the exception of collagen where some inhibition was seen at high Cx concentration (3.5 X 10(-3) M). Cx was also found to be ineffective at inhibiting L-[U-14C]leucine incorporation into platelet protein at all concentrations tested. These results suggest that the majority of platelet protein synthesis is mitochondrial and that this protein synthesis may have a role in human platelet aggregation.