It is important to assess the usefulness of long-term in vitro liver models for studying chronic toxicity, since acute assays may not reflect the in vivo situation. A potential long-term hepatocyte culture (i.e. liver spheroids) was investigated and compared to primary rat hepatocyte monolayer cultures following exposure to methotrexate (MTX), a well-documented chronic hepatotoxin. Following up to 7 days' treatment with MTX, cultures were morphologically assessed and assayed for enzyme leakage, intracellular reduced glutathione (GSH) and adenosine triphosphate (ATP). Spheroids maintained higher concentrations of GSH over the 14-day culture and ATP was maintained, but at a concentration not significantly different from monolayer cultures. Treatment of monolayer cultures resulted in concentration-related decreases in GSH and ATP, accompanied by enzyme leakage. In contrast, only ATP was affected following treatment of spheroids for 7 days. Spheroids appeared to be less sensitive to exposure to MTX, when compared with monolayer cultures. This may result from the maintenance of cellular functions, or from the lack of compound penetration into the three-dimensional spheroid structure. Therefore, the usefulness of spheroids to chronic in vitro toxicity testing may be limited.