The development of transgenic technology has enormous potential for livestock improvement. Several methods have been employed for generating transgenic livestock to date, the most popular being pronuclear injection or somatic cell nuclear transfer using genetically modified cells. However, the high cost and relatively low efficiencies seen with these methods have prompted development of alternative methods for producing transgenic livestock. Recently, the introduction of transgenes using viral vectors, in particular lentiviral vectors, has provided an avenue for increasing the efficiency of transgenic livestock production. Microinjection of concentrated lentivirus into the perivitelline space of oocytes and zygotes has been demonstrated as an effective means of creating transgenic livestock (cattle, sheep, and swine). In this study, we investigated the efficiency of producing transgenic sheep through microinjection of recombinant lentivirus into in vivo produced zygotes. Recombinant lentivirus was produced through co-transfection of HEK293T cells with various lentiviral transfer plasmids (each coding for a short hairpin RNA and a fluorescent marker protein) as well as a packaging plasmid and a plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), which was used to pseudotype viral particles. For lentivirus concentration, a total of 36 mL of viral supernatant was produced per viral construct. Viral supernatant was ultracentrifuged on a 15% sucrose cushion at 4°C for 1.5 h at a speed of 50 000 × g. The resulting viral pellet was resuspended in 30 μL of PBS and virus was frozen at –80°C until needed. Zygotes were surgically flushed from oviducts of superovulated donor ewes 24 h post-mating. Concentrated recombinant lentivirus (titer ≥ IU mL–1) was then microinjected into the perivitelline space. Injected embryos were surgically transferred into the oviduct of synchronized recipient ewes immediately after injection (3–4 embryos per ewe). Pregnancies were confirmed by ultrasound at 35 days of gestation. The pregnancy rate was 39% (45/114 ewes exhibiting at least one viable fetus). A subset of pregnant ewes was killed at 60 to 70 days gestation to harvest a total of 36 fetuses. Analysis of transgene incorporation was performed by PCR using genomic DNA isolated from skin and liver tissue samples. Two independent PCR reactions were performed per sample, and PCR analysis revealed 14 of the collected fetuses to be transgenic (39%). The remaining pregnancies were allowed to progress to term, and 32 lambs were born. Genomic DNA was isolated from blood samples collected on each lamb, and PCR analysis was performed as above. Of the lambs born, 13 of 32 (40%) were confirmed to be transgenic by PCR analysis. Southern blot analysis is currently underway to confirm PCR data. These results demonstrate that microinjection of recombinant lentivirus into in vivo produced sheep zygotes is an effective and efficient method for generating transgenic sheep.