Ornithine transcarbamylase (OTC) deficiency, the most common and severe inborn error of the urea cycle in humans, remains without adequate treatment, and mortality rates are high. Adenoviral vectors provide an efficient system for gene delivery, but there are problems, including toxicity. Efficient promoters that reduce the amount of vector required for treatment need to be developed. We constructed two recombinant adenoviral vectors, AdexCAGhOTC and AdexSR alpha hOTC, which harbor the human OTC gene under transcriptional control of CAG (a modified chicken beta-actin promoter with CMV-IE enhancer) and SR alpha (the SV40 early promoter with the R segment and part of the US segment of the HTLV-1 LTR), respectively. Each was tested in adult spf(ash) mice, an animal model of human OTC deficiency, and in primary human hepatocytes with OTC deficiency. Spf(ash) mice have a pronounced orotic aciduria as seen in humans. A complete recovery of hepatic OTC activity with minimal tissue damage was observed in these animals following the intravenous administration of AdexCAGhOTC alone. Western blot analysis confirmed hepatic OTC expression and normalization of orotic aciduria was evident for 60 days. Enzyme activities of primary human hepatocytes infected with AdexCAGhOTC were 10-40 times higher than those with AdexSR alpha hOTC. Thus, the adenoviral vector with an efficient promoter such as CAG, can be given further consideration for possible gene therapy in humans with OTC deficiency.