Intracellular localization and the heat-dependent redistribution of estrogen and progesterone in human endometrial cells have been investigated by a fluorescent steroid-antibody technique. The dispersed endometrial cells were incubated with 5 X 10(-8) M estradiol-17 beta and progesterone in TC medium 199 containing 10% calf serum for 1--3 hr at 4 degrees C or 37 degrees C. An indirect immunofluorescent technique using FITC-labeled anti-rabbit IgG and steroid antibodies raised from rabbits immunizing with estradiol-6-oxime-BSA and progesterone-3-oxime-BSA was applied to the smear specimens. In normal endometrial cells in both proliferative and secretory stages, specific fluorescences to estradiol and progesterone were generally observed in the cytoplasm after incubation with the steroids at 4 degrees C for 1 hr. When these cells were incubated with the steroids at 37 degrees C for 1 hr, cytoplasmic and predominant nuclear fluorescences were detected, whereas the 3 hr-incubation at 37 degrees C resulted in disappearance of cytoplasmic fluorescence, remaining nuclear fluorescence alone. These fluorescences were remarkedly eliminated when endometrial cells were incubated with diethylstilbestrol and R-5020 prior to the incubation with estradiol and progesterone, respectively. These results indicate that the fluorescent steroid-antibody technique used in this study enables us to visualize subcellular localization of estradiol and progesterone possibly bound to receptors in each endometrial cell.