Abstract Objective We have previously reported that directly injecting low-titer retroviral vector supernatant into pre-immune sheep fetuses resulted in the transfer and long-term expression of the bacterial Neo R gene within the hematopoietic system of these animals for over 5 years. In the present studies, we investigated whether using a higher titer vector would enable more efficient transduction and expression of the transgenes within the hematopoetic cells in sheep injected in utero. Materials and Methods Sixteen pre-immune sheep fetuses were injected intraperitoneally with the G1nBgSvNa8.1 helper-free retroviral vector supernatant encoding the bacterial Neo R and LacZ genes (titer: 1 × 10 7 cfu/mL). Results Over the 2-year time course of these studies, the presence and expression of the Neo R and LacZ genes were demonstrated in 12 of the 14 animals evaluated by several immunological and biochemical methods. Seven of the 12 sheep examined by flow cytometric analysis contained ≥ 6% transduced peripheral blood lymphocytes. Vector distribution was widespread without any detectable pathology. Importantly, PCR analyses and breeding experiments demonstrated that the germ line was not altered. Conclusions These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a developing fetus and thus achieving long-term expression of the transgenes within the recipient's hematopoietic cells. Furthermore, expression of the Neo R gene from these studies was higher than that reported in our previous study in which a lower titer vector was used.