The direct vector injection approach was used in the fetal sheep model of in utero gene therapy to determine the effects of the recipient gestational age on the efficacy and pattern of liver, lung, and brain transduction and transgene expression. The livers contained foci of transgene-expressing hepatocytes and demonstrated an inverse correlation between recipient age and hepatocyte transduction/transgene expression, with higher levels of gene transfer/expression early in gestation and lower levels late in gestation. Conversely, the percentage of transgene-expressing cells within the lungs of these same animals increased with gestational age, with the majority of transduction occurring in epithelium and fibroblasts. In contrast to the lung and liver, transgene-expressing cells within the brain were extremely limited at all gestational ages tested. Our results demonstrate that numerous nonhematopoietic cells within the liver and lung are transduced following direct injection of murine retroviral vectors into fetal sheep and suggest that the developmental stage of each organ at the time of injection may determine its susceptibility to in utero gene transfer and subsequent levels of transgene expression. Our results suggest that with further vector optimization this approach may be useful for treating diseases that involve the lung and liver early in development.