Differences in regulation of the accumulation of PtdIns(3,4)P2 versus that of PtdIns(3,4,5)P3 were noted in thrombin-stimulated human platelets. The rapid (within 20 s) response of PtdIns(3,4,5)P3 contrasted with a distinct lag in the accumulation of PtdIns(3,4)P2 that was followed by a pronounced increase by 90 s. The presence of 2.5 mM-CaCl2 further elevated PtdIns(3,4)P2 by 50-120%, but only at a late stage (after 90 s). Tetrapeptide RGDS (Arg-Gly-Asp-Ser), which blocks the interaction of ligands such as fibrinogen with platelet integrin alpha IIb beta 3 (GPIIb-IIIa), inhibited only the late-phase PtdIns(3,4)P2 accumulation that was associated with added Ca2+. Although stimulated tyrosine phosphorylation of platelet protein (total cell lysate) was altered by Ca2+ or RGDS, we could not identify any such proteins that were affected comparably to PtdIns(3,4)P2. In contrast to the PtdIns(3,4)P2 response, the accumulation of PtdIns(3,4,5)P3 was unaffected by Ca2+ or RGDS at any time.