Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that ∼ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca(2+) release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca(2+) influx also persisted in the presence of nitric oxide, delivered from spermine NONOate, or increased ectonucleotidase levels (apyrase). Surprisingly, apyrase was more effective than prostacyclin and NO at limiting secondary P2X1 activation. Dilution of platelets reduced the average extracellular ATP level without affecting the percentage contribution of P2X1 receptors to collagen-evoked Ca(2+) responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca(2+) mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.