The aim of this study was to examine the homocysteine effect on phospholipase Cγ2 (PLCγ2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLCγ2. The tyrosine kinases p60src and p72syk appeared to be involved upstream. Reactive oxygen species were increased in homocysteine treated platelets. Likely oxidative stress could prime the non receptor‐mediated tyrosine kinase p60src, inducing phosphorylation and activation of p72syk. The antioxidant N‐acetyl‐L‐cysteine prevented the activation of these kinases. The phosphorylation and activation of PLCγ2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLCγ2, suggesting that thromboxane A₂ could be involved. In addition the treatment of platelets with homocysteine caused intracellular calcium rise and protein kinase C activation. Finally homocysteine induced platelet aggregation, that was partially reduced by indomethacin and by N‐acetyl‐L‐cysteine of 35% or 50% respectively, while the PLCγ2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLCγ2 plays an important role in platelet activation by homocysteine and that the stimulation of this pathway requires signals through oxygen free radicals and thromboxane A₂. J. Cell. Biochem. 100: 1255–1265, 2007. © 2006 Wiley‐Liss, Inc.