The fluorescent calcium indicator, fura 2, was used to test whether contraction of primary cultured smooth muscle cells (SMC) from small pulmonary arteries in response to hypoxia and the relaxation by large pulmonary and cerebral artery SMC were mediated by changes in cytoplasmic free Ca2+ (Ca2+c). Because SMC from large pulmonary and cerebral arteries contract to norepinephrine (NE), Ca2+c levels during NE exposure were measured to determine if they differed from those seen with hypoxia. Under hypoxic conditions, Ca2+c increased 64.1 +/- 11.1% above the normoxic baseline in small pulmonary artery SMC. In SMC from large pulmonary and cerebral arteries, Ca2+c decreased 25.2 +/- 9.20 and 28.1 +/- 5.80%. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and ryanodine attenuated the Ca2+c increase in the small pulmonary artery SMC. On exposure to NE, Ca2+c increased markedly in all three SMC types. EGTA and ryanodine treatment also attenuated NE-induced Ca2+c increases in all three SMC types. These results show that the three SMC types mobilize their available Ca2+ stores differently when exposed to hypoxia but similarly when exposed to NE. The data also suggest that a change in the Ca2+c concentration, rather than a change in the Ca2+ sensitivity of the contractile apparatus, is involved in the response to hypoxia.