Previous studies indicate that in pulmonary and coronary arterial smooth muscle cells beside IP3 and ryanodine sensitive Ca2+ pools, acid-filled Ca2+ store may play an important role in vasoconstriction. The aim of this study is to characterize the Ca2+ pools involved in phenylephrine (PHE) stimulation of rat aortic smooth muscle cells. Experiments were carried out using Ca2+-imaging technique on cultured rat aortic smooth muscle cells and force contraction of desendothelized rat aortic ring. Administration of PHE (1 microM) in Ca2+ free saline on cultured aortic smooth muscle cells induces a transitory elevation of [Ca2+]i by 561 + 3.6 nM (n = 38 cells). Following one hour pretreatment with bafilomycin (1 microM), a well known disrupter of acid-filled Ca2+ stores, the [Ca2+]i diminished up to 174 + 2.8 nM (n = 47 cells). The blockade of ryanodine receptors with ryanodine (10 microM; 15 min preincubation), or the IP3 receptors with 2-APB (2-aminoethyl diphenylborate – 100 microM; 15 min preincubation) also reduce the PHE-induced elevated [Ca2+]i up to a level of 352 + 2.9 nM (n = 26) and 231 + 2.3 nM (n = 34), respectively. Meanwhile 1 h pretreatment with bafilomycin (1 microM) and 15 min with 2-APB (100 microM) totally abolished the elevation of cytoplasmatic Ca2+ induced by PHE (n = 52). Experiments carried out on desendothelized rat aorta rings reveal that 1 h treatment with bafilomycin (1 microM) inhibits the PHE-induced contraction with 58 + 3.2% (n = 6), while 45 min treatment with ryanodine (10 microM) or 2-APB (100 microM) reduce the contractions elicited by PHE with 31 + 2.8% (n=6) and 47 + 3.6% (n = 6), respectively. Concomitant treatment with bafilomycin (1 microM; 1 h), and 2-APB (100 microM; 45 min) totally abolished PHE-induced contraction (n = 6). It may be thus concluded that PHE primary recruits Ca2+ from acid-filled and IP3-sensitive Ca2+ stores which, in turn, activate ryanodine receptors through calcium-induced calcium-release mechanism.