The Oct4 gene is an essential transcription factor for maintenance of pluripotency in mammals. Here, we report the production of cloned transgenic pigs carrying a genomic construct encompassing murine Oct4 regulatory regions and driving an enhanced green fluorescent protein (Oct4-EGFP) construct. We employed fetal porcine fibroblasts, stably co-transfected with neomycin and the mouse Oct4-EGFP construct, for somatic cell nuclear transfer to reconstruct transgenic embryos. The cloned embryos (811 embryos) were surgically transferred into the oviducts of 8 recipient animals. Two pregnancies were terminated at Day 25 for recovery of fetuses and the others delivered a total of 23 piglets, of which 11 survived the postpartum period. A detailed analysis showed that the Oct4-EGFP construct was active in cloned pig blastocysts from Days 5 to 6. EGFP fluorescence was found exclusively in the primordial germ cells of Day 25 fetuses, whereas somatic tissues did not express the transgene. We could also detect expression of Oct4-EGFP in individual cells of the postnatal testis. Testis-specific expression was confirmed by Northern blotting. We fused transgenic porcine fibroblasts with murine embryonic stem cells to analyze reactivation of the Oct4-EGFP transgene under experimental reprogramming conditions. The fused hybrids displayed stem cell morphology and a high proliferation rate and started to express EGFP fluorescence 72 h after fusion. In conclusion, we report the production of viable Oct4-EGFP transgenic piglets that express EGFP exclusively in germ line and pluripotent cells. This transgenic pig line is a valuable tool for derivation and maintenance of porcine embryonic stem cells and will be of utmost interest for reprogramming studies and for preclinical testing of stem cell therapies in a large animal model.