Homocysteine (Hcy) induces matrix metalloproteinase-9 (MMP-9) in microvascular endothelial cells (MVEC). We hypothesized that the extracellular signal-regulated protein kinase-1/2 (ERK-1/2) signaling pathway is involved in Hcy mediated MMP-9 expression. In cultured MVEC, Hcy induced activation of ERK which was blocked by PD98059 and U0126 (MAPK kinase inhibitors). Pretreatment with acetoxymethyl)-1, 2-bis (o-aminophenoxy) ethane N, N, N', N'-tetraacetic acid (BAPTA-AM), staurosporine (protein kinase C (PKC) inhibitor), or Go6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of calcium and calcium-dependent PKC in Hcy induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G-protein coupled receptors (GPCR) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVEC with genestein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting involvement of protein tyrosine kinase pathway in Hcy induced ERK activation which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by pre-incubation with n-acetylcysteine (NAC), and superoxide dismutase suggesting the role of oxidative in Hcy-induced ERK activation. Pretreatment with ERK-1/2 blocker (PD98059), staurosporine, folate, or NAC modulated the Hcy induced MMP-9 activation as measured by zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK-1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVEC.