The generation of a large number of fully functional hepatocytes from a renewable cell source can provide an unlimited resource for bioartificial liver devices and cell replacement therapies. We have established a directed differentiation system using sodium butyrate treatment to generate an enriched population of hepatocyte-like cells from embryonic stem cells. A metabolic analysis of the hepatocyte populations revealed glycolytic and mitochondrial phenotypes similar to mouse hepatoma cells, implying that these cells represent an immature hepatocyte phenotype. To mediate further differentiation, S-NitrosoAcetylPenicillamine (SNAP), a nitric oxide donor, was utilized to induce mitochondrial development in the precursor populations. A comparative analysis of the different treated populations showed that 500microM SNAP treatment resulted in the generation of an enriched population of metabolically mature hepatocyte-like cells with increased differentiated function. Specifically, 500microM SNAP treatment increased glucose consumption, lactate production rates, mitochondrial mass, and potential as compared to untreated populations. In addition, functional analysis revealed that intracellular albumin content, urea secretion rates, and cytochrome P450 7a1 promoter activity were increased in the treated population. The methodology described here to generate an enriched population of metabolically and functionally mature hepatocyte-like cells may have potential implications in drug discovery and regenerative medicine.