Cultured cells are dramatically affected by the micro-environment in which they are grown. In this study, we have investigated whether HepG2 liver cells grown in three dimensional (3-D) cultures cope more effectively with the known cytotoxic agent, methotrexate, than their counterparts grown on traditional two dimensional (2-D) flat plastic surfaces. To enable 3-D growth of HepG2 cells in vitro, we cultured cells on 3-D porous polystyrene scaffolds previously developed in our laboratories. HepG2 cells grown in 3-D displayed excellent morphological characteristics and formed numerous bile canaliculi that were seldom seen in cultures grown on 2-D surfaces. The function of liver cells grown on 3-D supports was significantly enhanced compared to activity of cells grown on 2-D standard plasticware. Unlike their 2-D counterparts, 3-D cultures were less susceptible to lower concentrations of methotrexate. Cells grown in 3-D maintained their structural integrity, possessed greater viability, were less susceptible to cell death at higher levels of the cytotoxin compared to 2-D cultures, and appeared to respond to the drug in a manner more comparable to its known activity in vivo. Our results suggest that hepatotoxicity testing using 3-D cultures might be more likely to reflect true physiological responses to cytotoxic compounds than existing models that rely on 2-D culture systems. This technology has potential applications for toxicity testing and drug screening.