Objective: We sought to examine the change of cytosolic calcium concentration caused by prostaglandin F_2α and RU 486 in cultured human myometrial cells. Study Design: Human myometrial cells obtained from 16 nonpregnant women were loaded with fura 2, and the intracellular cytosolic calcium concentrations were measured by the use of wavelength spectrophotofluorometry. Results: Application of prostaglandin F_2α (2.8 μmol/L) caused an initial rapid rise in cytosolic calcium concentration followed by sustained cytosolic calcium oscillations at an average frequency of 0.43 ± 0.04 min^–1 and an amplitude in the range of 296.82 ± 27.16 nmol/L. The oscillatory activity was not affected by increasing the concentration of prostaglandin F_2α but varied by changing the concentration of extracellular cytosolic calcium concentration. The cytosolic calcium oscillations were suppressed by caffeine, 2,5-di-tert-butylhydroquinone, and lanthanum but not affected by ryanodine. Verapamil decreased the amplitude but not the frequency of oscillations. The progesterone antagonist RU 486 at a concentration of 10^–8 to 10^–5 mol/L had no significant effect on the basal intracellular cytosolic calcium. However, RU 486 (10^–5 mol/L) significantly increased the frequency but not the amplitude of intracellular cytosolic calcium oscillations induced by prostaglandin F_2α. Conclusion: The results indicate that prostaglandin F_2α–stimulated cytosolic calcium oscillations are mediated by an increase in both cytosolic calcium release from inositol 1,4,5-trisphosphate–sensitive cytosolic calcium stores and a cytosolic calcium influx from the extracellular space. Moreover, RU 486 seems to directly regulate prostaglandin F_2α–induced intracellular cytosolic calcium in human myometrial cells. (Am J Obstet Gynecol 2000;182:582-8.)