Thrombospondin-1 (TSP1), a multifunctional extracellular matrix glycoprotein, has been shown to suppress the angiogenic response in vivo and in vitro. We hypothesized that TSP1 might play a role in the inhibition of capillary morphogenesis during the endometrial cycle and examined its expression in 46 human endometrial specimens. Our results show that the expression of TSP1 in the endometrium is (a) cycle-dependent, (b) associated with periods of low capillary growth, and (c) regulated by progesterone. TSP1 protein was identified in the basement membrane of capillaries of the functional endometrium during the secretory phase. Abundant expression of TSP1 mRNA in the secretory phase was also detected by in situ hybridization, in contrast to the low levels seen in the proliferative phase. These findings were confirmed by Northern analysis of proliferative and secretory endometrium. Transcripts for TSP1 were observed predominantly in stromal cells, but signal was also detected in some endothelial and smooth muscle cells. Since the proliferation of endometrial tissue is regulated by steroid hormones, we tested the effects of estrogen and progesterone on TSP1 expression by stromal cells isolated from human endometrium. We found that levels of TSP1 mRNA and protein were increased after incubation with progesterone. Maximal stimulation of mRNA was observed after 8 h of treatment with 10-50 microM progesterone, and the effect was suppressed by the progesterone antagonist RU-486. Induction by progesterone was cell-specific and equivalent to the stimulation mediated by PDGF. Finally, the levels of TSP1 present in progesterone-stimulated cultures were sufficient to inhibit the migration of endothelial cells in vitro; this effect was nullified by anti-TSP antibodies. We therefore propose that the production of TSP1 at later stages of the endometrial cycle is linked to the inhibition of vessel formation and that TSP1 expression is progesterone-dependent in this tissue.