The present study was designed to investigate the mechanism of action of low extracellular magnesium ion concentration ([Mg²⁺]_o) on isolated canine basilar arteries and single cerebral vascular smooth muscle cells from these arteries. Low-[Mg²⁺]_o medium (0-0.6 mM) produces endothelium-independent contractions in isolated canine basilar arteries in a concentration-dependent manner; the lower the concentration of [Mg²⁺]_o, the stronger the contractions. The low-[Mg²⁺]_o medium-induced contractions are significantly attenuated by pretreatment of the arteries with low concentrations of either SB-203580, U-0126, PD-98059, genistein, or an Src homology 2 (SH2) domain inhibitor peptide. IC₅₀ levels obtained for these five antagonists are consistent with reported inhibitor constant (K_i) values for these tyrosine kinase and mitogen-activated protein kinase (MAPK) antagonists. Low-[Mg²⁺]_o medium (0-0.6 mM) produces transient intracellular calcium ion concentration ([Ca²⁺]_i) peaks followed by a slow, sustained, and elevated plateau of [Ca²⁺]_i in primary single smooth muscle cells from canine basilar arteries. Low-[Mg²⁺]_o medium induces rapid and stable increases in [Ca²⁺]_i; these increases are inhibited markedly in the presence of either SB-203580, U-0126, PD-98059, genistein or a SH2 domain inhibitor peptide. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the low-[Mg²⁺]_o-induced contractions or the elevation of [Ca²⁺]_i. The present study suggests that activation of several cellular signaling pathways, such as protein tyrosine kinases (including the Src family) and MAPK, appears to play important roles in low-[Mg²⁺]_o-induced contractions and the elevation of [Ca²⁺]_i in smooth muscle cells from canine basilar arteries.