An organ culture technique for the maintenance of human endometrium was used to study secretory protein production and the ability of progesterone to alter the character of secretory products from this tissue. Proliferative phase cultures of human endometrium were incubated in defined medium for 48 h in the presence or absence of 0.1 μg/ml progesterone. During the 25th to 48th hours, the tissues were labeled with radioactive protein precursors. The proteins in medium and tissue cytosol were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. Our results suggest that human endometrium secretes at least 24 proteins into the culture medium and that the majority of these are glycoproteins. The presence of progesterone during culture caused a change in the concentration of certain medium proteins. Five protein bands were consistently observed to have a hormonally induced variation of intensity on autoradiographs. Two of these (mol wt, 58,000 and 28,000) showed a decrease in their intensity and three (mol wt, 130,000, 50,000, and 35,000) showed an increase when progesterone-treated and untreated cultures of endometrium were compared. The greatest progesteroneinduced change in intensity occurred with the 58,000 mol wt protein (S2). The changes in band intensity appear to be reversible over the time periods studied. Our results provide evidence that significant alterations occur in the protein content of human endometrial secretions as a result of progesterone stimulation.