An increase in intracellular calcium is the major trigger for contraction in vascular smooth muscle. However, the exact mechanisms that control Ca²⁺ homeostasis, in particular Ca2+ entry mechanisms, still remain to be fully elucidated. Many studies have manipulated extracellular calcium in order to investigate these mechanisms (Robertson et al., 2000; Ng & Gurney, 2001; Snetkov et al., 2003). Here we present data showing the activation of a voltage independent, non-selective cation current (termed I_C) in pulmonary artery (PA) and mesenteric artery (MA) smooth muscle cells (SMCs) upon removal of extracellular Ca²⁺ (Ca²⁺_o) and in the presence of 1 mM EGTA. Male Wistar rats (225-300 g) were humanely killed, and small intra-pulmonary (<500 μm) and mesenteric (<300 µm) arteries were dissected. Cells were isolated as previously described (Smirnov et al., 2002). Experiments were performed at room temperature, with a Cs⁺ and low Cl^–-based pipette solution, in the perforated patch mode of the voltage clamp-technique (100 µg/ml amphotericin B). The external solution contained (mM): 130 NaCl, 5 CsCl, 1.2 MgCl₂, 1.5 CaCl₂, 10 HEPES, 10 glucose (pH=7.2). 4-aminopyridine and TEA (both at 5 mM) were added to block Cs⁺ flux through K⁺ channels. In PA SMCs, I_C was observed with greater frequency than in MA (152 of 160 cells in PA; 35 out of 54 cells in MA)and was larger in amplitude (with the mean current density at -100 mV equal to 97.8±7.8 pA/pF in PA and 50.1 ± 9.9 pA/pF in MA, p < 0.05). In both tissues, I_C was significantly blocked by lanthanum (100 µM), gadolinium and cadmium (100 µM); nickel (100 µM) also significantly reduced I_C in PA SMCs (by 32.3±13.9% at -100 mV, n=7; p < 0.05). Biophysical characterisation of I_C in PA SMCs suggests that I_C exhibited the following selectivity sequence Cs⁺≤Rb⁺=K⁺=Na⁺≤Li⁺ < NMDG (n ≤ 8), suggesting weak binding within the channel pore. Ca²⁺_o sensitivity, assessed in PA SMCs, showed that I_C was blocked by 1 µM Ca²⁺_o by 74.5±8.8% at -100mV (n=5). In PA SMCs, I_C activation was not significantly affected by cell pre-treatment with 1 µM thapsigargin, whereas in MA SMCs, its activation was significantly enhanced. Qualitatively similar results were observed with 10 µM cyclopiazonic acid. These results suggest that activation of I_C is functionally coupled to depletion of intracellular Ca²⁺ stores in MAs, but not in PAs. Although, due to its high Ca²⁺_o sensitivity, the functional role of I_C is not currently clear, activation of I_C by intracellular or extracellular, yet to be identified, factors, cannot be excluded.