Verapamil at a concentration of 10(-4) M inhibited aggregation and release of [3H]5HT induced by platelet activating factor (PAF-acether, PAF) in rabbit platelet-rich plasma and washed labelled platelets. When added to platelets previously aggregated by PAF-acether verapamil caused them to desaggregate at doses as low as 2 X 10(-6) M. The desaggregated platelets were refractory to further additions of similar doses of PAF-acether but could further be aggregated by A23187. Simultaneous to full aggregation PAF-acether caused phosphorylation of 40K and 20K proteins in particular. Addition of verapamil at the concentration of 2 X 10(-6) M to platelets already aggregated by PAF-acether resulted in dephosphorylation of 40K protein and reduction of phosphorylation of 20K protein to the level of control parallel to desaggregation. TMB-8 (10(-3) M) also caused desaggregation and reversal of phosphorylation of 40K and 20K proteins. When A23187 was added to verapamil desaggregated platelets, 40K and 20K proteins were rephosphorylated. The extracellular calcium antagonists EGTA or La3+, when added to PAF-acether aggregated platelets, did not abolish the phosphorylation of 40K and 20K proteins. The experiments suggest that inhibition of intracellular calcium-dependent reactions is involved in the desaggregatory action of verapamil.