Term decidual tissue consists of terminally differentiated decidual cells, bone marrow-derived cells. and fibroblast cells. Since undifferentiated decidual cells are fibroblast-like cells of the endometrial stroma, the possibility exists that the fibroblast cells in term decidua are undifferentiated decidual cells. To test this hypothesis, a purified population of fibroblast cells was isolated from term decidua and treated under conditions that are known to induce differentiation of endometrial stromal cells. By flow cytometry and immunocytochemistry, the fibroblast cells from term decidua were shown to be free of cells expressing bone marrow-derived cell-surface antigens and the epithelial cell marker cytokeratin. In addition, they tested positive for the cytoskeletal protein vimentin, thus establishing that they were mesenchymal cells. As with endometrial stromal cells, continuous treatment of the decidual fibroblast cells with the progesterone analog medroxyprogesterone acetate and estradiol in combination with either dibutyryl-cAMP or prostaglandin E2 induced cell aggregation and the expression of prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). When cells were plated at an initial cell density of 0.25 x 106 cells/well in a 24-well culture dish with medium changes every three days, PRL was first detected on Days 4-6, and the peak of averaged 24 h-PRL release (30 ng/well) occurred on Days 26-28. The mRNA for decidual fibroblast PRL followed a temporal pattern corresponding to that of the released hormone. The size of the PRL mRNA was 1.15 kb, corresponding to the alternately spliced PRL mRNA reported for decidualized endometrial stromal cells and other extrapituitary sources of PRL. In contrast to PRL release, IGFBP-I release was first observed during treatment Days 1-3, and high levels of IGFBP-1 were then released into the medium from Day 4 through at least Day 12. A significant decrease in IGFBP-1 release was first observed during treatment Days 17-19, and the levels of IGFBP-1 then gradually decreased. Release of IGFBP-1 was accompanied by a corresponding increase in IGFBP-1 mRNA. However, whereas the protein levels diminished by Day 19, the mRNA levels appeared to remain elevated until Day 24. The changes in morphology and the induction of PRL and IGFBP-1 expression in decidual fibroblast cells closely resembled the response of endometrial stromal cells cultured under similar conditions. The results suggest that the decidualized uterine endometrium maintains a proliferating population of cells that can be recruited for differentiation to the decidual cell phenotype throughout pregnancy.