The aim of this study was to develop immortalized cell lines from porcine uterus. Endometrial cells including luminal epithelium (LE), glandular epithelium (GE), stroma (ST), and myometrium (MYO) were enzymatically isolated from the uterus of a day 12 pregnant gilt. Primary cultures were immortalized by transduction with a retroviral vector containing the E6 and E7 open reading frames of human papillomavirus type 16 (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analog G418 (0.4–1.5 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for 1 yr. Expression of the E7 protein was confirmed in all cell lines by Western blotting. Phase contrast microscopy revealed that LE and GE cells exhibited cobblestone morphology, whereas ST and MYO cells exhibited spindle-shaped morphology. The epithelial origin of LE and GE was confirmed by positive immunostaining for cytokeratin. Stromal and MYO cells were vimentin-positive, but cytokeratin-negative. The MYO cell lines were positive for smooth muscle α-actin staining, whereas LE, GE, and ST cell lines were negative for α-actin. Western blotting indicated that all cell lines expressed both estrogen and progesterone receptors, but only GE cells secreted uteroferrin (UF). Collectively, these porcine uterine cell lines provide an in vitro model for studying cell type–specific actions of hormones and cytokines, signal transduction pathways, cell–cell interactions, and gene expression.