Slowly hydrolysable analogues of GTP were introduced into hepatocytes by incubating the cells in the absence of Mg2+ and in the presence of ATP4-. Experiments using guanosine 5'-[γ-[35S]thio]triphosphate (GTP[35S])indicated that about 50% of the GTP[S] loaded into the cells was subsequent hydrolysed. In cells loaded with GTP[S] and incubated in the absence of added extracellular Ca2+ (Ca2+0), the rate of activation of glycogen phosphorylase observed after addition of 1.3 mM-Ca2+ was 250% greater than the rate observed in unloaded cells. Smaller effects (130%) were observed in cells loaded with either guanyl-5'-yl imidodiphosphate or guanosine 5-[β-thio]diphosphate (GDP[S]). Cells loaded with adenosine 5'-[γ-thio]triphosphate showed no increase in glycogen phosphorylase activity on addition of Ca2+0. The effect of a submaximal concentration of GTP[S] on the Ca2+-induced activation of glycogen phosphorylase was additive with that of a half-maximally effective concentration of vasopressin. GTP[S] did not increase the effect of a maximally effective concentration of the hormone. Cells loaded with GTP[S] exhibited an increased initial rate of 45Ca2+ exchange measured at 1.3 mM-Ca2+0. GTP[S] did not affect the amount of 45Ca2+ exchanged by cells incubated at 0.1 mM-Ca2+0 or the ability of vasopressin to release 45Ca2+ from these cells. It is concluded that the introduction of slowly hydrolysable analogues of GTP to the liver cell cytoplasmic space stimulates the inflow of Ca2+ across the plasma membrane through a channel similar to that activated by vasopressin.