Vaginal and uterine stromal (VS and UtS) cells have been cultured in a collagen gel matrix, and the ability of the cells to retain their identity and interact normally with epithelia after culture was examined. Stromal explant from 2-d-old mice were plated onto an extracellular matrix covered with collagen, and maintained in Ham’s F12∶DMEM (1∶1) containing 15% fetal bovine serum. The fibroblastic stromal cells invaded and eventually filled the overlying collagen during the 4-wk growth period, and the total DNA of the UtS and VS cultures increased 3.5- and 4-fold, respectively. To assess the ability of the cultured stroma to perform its normal functions after the in vitro period, recombinations of cultured stroma and fresh epithelia were preparaed and transplanted under the renal capsule of female hosts and grown for 4 wk. The epithelium in recombinants of cultured VS + vaginal epithelium (VE) and cultured UtS + uterine epithelium (UtE) was histologically normal and proliferated in response to estrogen. Cultured stroma also instructively induced heterologous epithelium; VS induced UtE to undergo vaginal differentiation, and UtS induced VE to undergo uterine differentiation. These results indicate that UtS and VS retain their identity and do not irreversibly dedifferentiate in culture. Stromal cells grown in a colagen gel matrix form a functional stroma; they interact normally with epithelium after culture and express normal permissive and instructive inductive functions when reassociated with epithelium and grown in vivo.