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Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.