Summary Synthesis of D-3-phosphorylated phosphoinositides and its correlation with protein-tyrosine phosphorylation were examined using human platelets. Thrombin stimulation of platelets resulted in time- and dose-dependent production of phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P 2), which is absent from resting platelets. In contrast, phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P 3) was detected in resting platelets, but remained unaffected by thrombin treatment. The production of Ptdlns(3,4)P 2 but not Ptdlns(3,4,5)P 3 was inhibited by pretreatment with staurosporine or dibutyryl cyclic adenosine monophosphate (dbcAMP). Protein-tyrosine phosphorylation, which is reportedly involved in generation of 3-phosphorylated phosphoinositides, was elicited in thrombin-activated platelets. The tyrosine phosphorylation was suppressed by pretreatment with staurosporine or dbcAMP. These observations suggest that synthesis of Ptdlns(3,4)P 2 but not Ptdlns(3,4,5)P 3 is closely correlated with protein-tyrosine phosphorylation in human platelets.