Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.