The effect of progestin on the rate of estradiol (E2) sulfurylation in human endometrium was studied in two culture systems: 1) in intact endometrial tissue fragments and 2) in isolated endometrial epithelial glands and stromal cells. Human endometrial tissue fragments were cultured in medium in the presence and absence of progesterone (P) for 24-48 h. The arylsulfotransferase activity was determined in the cytosol of the tissue homogenate by measuring the rate of conversion of E2 to E2-3 sulfate (E2S) in the presence of an excess amount of substrate and cofactor (adenosine-3'-phosphate-5'-phosphosulfate). The enzyme activity was found to be stimulated in the sample cultured with P. The increase in activity correlated with the dose of P and period of culture. Stimulation of endometrial estradiol dehydrogenase activity by P, which has been reported in a series of previous publications, was also evident in the current study. Human endometrial glandular epithelial and stromal cells were cultured separately in medium in the presence and absence of medroxyprogesterone acetate (MPA). The cultured cells were incubated with E2 (0.4-0.6 nM) for 2 h. The formation of estrogen sulfates, [estrone sulfate (E1S) and E2S] was greatly increased in all the proliferative glandular epithelial cells cultured with MPA. However, the rate of sulfurylation was not appreciably affected by MPA in stromal cells and in glandular epithelial cells from secretory endometrium. The oxidation of E2 in these cultured cells was also influenced by P but not to the extent of sulfurylation. These results indicate that arylsulfotransferase in endometrium originates from glandular epithelial cells and can be stimulated by progestin.