The hormonal effects of prostaglandin F₂ (PGF₂) in luteal cells include mobilization of intracellular calcium ([Ca]i), generation of reactive oxygen species (ROS), depletion of luteal ascorbate levels, inhibition of steroidogenesis, and ultimately cell death. In the present paper, we investigate the hypothesis that [Ca]i mobilization stimulates the generation of ROS, that results in depletion of cellular ascorbic acid in rat luteal cell, and results in luteolysis and cell death. We used fluorescence microscopy and fluorescent probes (fura-2 and dichlorofluorescein diacetate) to measure [Ca]i and ROS, respectively, in single cells. We also used a self-referencing ascorbic acid-selective electrode that noninvasively measures ascorbic acid flux at the extended boundary layer of single cells. Menadione, a generator of intracellular superoxide radical (O₂), PGF₂, and calcium ionophore increased [Ca]i, and stimulated the generation of intracellular ROS. In the case of calcium ionophore and PGF₂, but not menadione, the generation of ROS was dependent on extracellular calcium influx. We measured ascorbic acid flux in unstimulated cells and found a net efflux of 121.5 ± 20.3 fmoles.cm^{- 1}.s^-1 (mean ± SEM, n=8), but in the absence of xtracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmoles.cm^-1.s^-1 n=5, p < 0.05). PGF₂ and menadione stimulated ascorbic acid efflux, but calcium ionophore had no significant effect. These data suggest two regulatory mechanisms of ascorbic acid in luteal cells. Under basal conditions, a calcium-dependent efflux is present, that may represent recycling and maintenance of an antioxidant gradient of ascorbic acid at the plasma membrane. Under the influence of luteolytic hormone and /or oxidative stress, ascorbic acid efflux is stimulated, which does not appear to be dependent on extracellular calcium influx, or generation of ROS. Although sitespecific mobilization of calcium pools and formation of ROS cannot be ruled out, the release of ascorbate by PGF₂-stimulated luteal cells may occur through some other signaling pathway.