Experiments were undertaken to investigate the regulation of capacitative Ca2+ entry by phorbol ester-sensitive protein kinase C and serine/threonine protein phosphatase activity. The thapsigargin-activated Ca2+ entry pathway was probed in control cells and cells treated with phosphatase type 1/2A inhibitors, okadaic acid and calyculin A, or with the phorbol ester, phorbol 12-myristate 13-acetate. The permeability state of this pathway was monitored in the presence or absence of these agents using fluorometric measurements of intracellular Ca2+ concentration, unidirectional Mn2+ entry, and membrane potential and unidirectional measurements of Ca2+ uptake using 45Ca2+. The results of these studies demonstrate that modification of the phosphorylation state of target protein(s) on serine/threonine amino acid residues by inhibition of phosphatase type 1/2A inhibits the capacitative Ca2+ entry pathway in rat thymic lymphocytes. Importantly, the capacitative Ca2+ entry pathway in rat thymic lymphocytes is not modulated by activation of phorbol ester-sensitive protein kinase C.