The in vitro evaluation of hepatotoxicity is an essential stage in the research and development of new pharmaceuticals as the liver is one of the most commonly impacted organs during preclinical toxicity studies. Fresh primary hepatocytes in monolayer culture are the most commonly used in vitro model of the liver but often exhibit limited viability and/or reduction or loss of important liver-specific functions. These limitations could potentially be overcome using three-dimensional (3D) culture systems, but their experimental nature and limited use in liver toxicity screening and drug metabolism has impaired their uptake into commercial screening programs. In this study we use a commercially available polystyrene scaffold developed for routine 3D cell culture to maintain primary rat hepatocytes for use in metabolism and toxicity studies over 72 h. We show that primary hepatocytes retain their natural cuboidal morphology with significantly higher viability (>74%) than cells grown in monolayer culture (maximum of 57%). Hepatocytes in the 3D scaffolds exhibit differential expression of genes associated with phase I, II, and III drug metabolism under basal conditions compared with monolayer culture and can be induced to stably express significantly higher levels of the cytochrome-P450 enzymes 1A2, 2B1, and 3A2 over 48 h. In toxicity studies the hepatocytes in the 3D scaffolds also show increased sensitivity to the model toxicant acetaminophen. These improvements over monolayer culture and the availability of this new easy to use 3D scaffold system could facilitate the uptake of 3D technologies into routine drug screening programs.