The present study was designed to investigate the direct response of fetal adrenomedullary cells to hypoxia, and the possible change in this responsiveness with maturation. Ovine fetal adrenomedullary cells, when exposed to 30 min of hypoxia induced by perfusing with Krebs-Henseleit solution equilibrated with 1% O2, released significantly greater amounts of total catecholamine into the perfusate, compared to basal conditions. After a 1-h control period, a second 30-min hypoxic episode stimulated a catecholamine response which was significantly smaller in magnitude than the first. Following the two hypoxic episodes, the cells were capable of responding to 50 mM KCl with a large increase in total catecholamine release. During the first hypoxic episode, the release of both norepinephrine and epinephrine was stimulated by equal magnitude. Fetal adrenomedullary cells obtained from fetuses at 100, 120, and 130 days gestation showed similar responsiveness to the same hypoxic stimulus, and these responses were not different from that observed in maternal adrenomedullary cells. On the contrary, responsiveness to KCl-induced depolarization was greatest in cells obtained from fetuses at 130 days gestation when compared to that in the younger fetuses. This increased responsiveness to KCl was accompanied by a greater catecholamine store in the adrenal medulla of the fetuses at this gestational age. These results suggest that ovine fetal adrenomedullary cells can respond directly to hypoxia by releasing catecholamines. This direct responsiveness became desensitized after repeated exposure. Finally, a decrease in direct responsiveness to hypoxia associated with maturation could be demonstrated.