Agonist-stimulated Ca2+ influx is critically important to mediate the function of endothelial cells. It has been suggested that release of Ca2+ from internal stores activates Ca2+ influx across the plasma membrane. In the present study, we investigated the effects of ML-9, a myosin light-chain kinase (MLCK) inhibitor, and genistein, a tyrosine kinase inhibitor, on the agonist stimulated Ca2+ response in porcine aortic endothelial cells loaded with a Ca2+-sensitive dye, fura-2. ML-9 almost completely abolished Ca2+ influx, whereas genistein only partially attenuated Ca2+ entry. Both of them did not affect the mobilization of Ca2+ from internal stores. In contrast, genistein was more potent in the inhibition of Mn2+ influx than ML-9. These findings indicate the different selectivity for Ca2+ and Mn2+ in the cation entry pathway in agonist-stimulated endothelial cells.