Thrombin has been shown to be involved in abnormal proliferation and pathogenic vascular remodeling associated with cardiovascular pathologies including atherosclerosis. The objective of this study was to determine the modulation by Ca2+ ions of p42/p44 MAP kinase (ERK1/2) signaling pathway triggered by thrombin in vascular smooth muscle cells (VSMCs). Experiments were performed in vitro with cultured VSMCs isolated from rat thoracic aorta. Since the stimulation of VSMC by thrombin results in the sequential phosphorylation/activation of Src, epidermal growth factor receptor (EGFR) and ERK1/2, the influence of Ca2+ ions on thrombin-induced phosphorylation of these signaling proteins was investigated. Antibodies directed against the phosphorylatyed, hence activated forms, of kinases of Src family (Tyr416), EGFR (Tyr1068) and ERK1/2 (Thr202, Tyr204) were used. PP2 and GF109203X were used as inhibitors of Src family kinase and protein kinase C (PKC), respectively; AG1478 and PD153035 were used as inhibitors of EGFR kinase; U0126 and PD98059 were used as inhibitors of ERK1/2 kinase (MEK). Intracellular Ca2+ concentration was measured using the Fura-2 technique. The roles of intracellular Ca2+ concentration ([Ca2+]i) and of Ca2+ mobilization were investigated with Ca2+ chelator BAPTA-AM and thapsigargin, respectively. Immunoblotting of proteins allowed us to evaluate their phosphorylation/activation status. Under physiological conditions, results showed that Src and Pyk2 activities were involved upstream of EGFR kinase transactivation that was required for ERK1/2 activation. However, blunting [Ca2+]i elevation with BAPTA abolished thrombin- and thapsigargin-induced EGFR transactivation but did not affect ERK1/2 activation. Under such conditions, the inhibition of EGFR kinase with AG1478 or PD153035 did not affect significantly ERK1/2 activation but the addition of PP2 did inhibit the activation of all signaling proteins understudied. Results also showed that the inhibition of EGFR and ERK1/2 activation by PKC inactivation with GF109203X, could be overcome by pretreating VSMC with BAPTA-AM. This indicated that upon thrombin stimulation, ERK1/2 activation could dissociate from EGFR activation and suggested the existence of a new signaling pathway whereby ERK1/2 could be activated on a Src- and MEK-dependent but EGFR- and PKC-independent manner. This new pathway that is unmasked by the prevention of [Ca2+]i elevation, can be of importance under pathological circumstances which inerfere with calcium homeostasis and particularly in cardiovascular therapy since most of the drugs used are known to affect the intracellular [Ca2+] concentration.