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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 …