The toxicity of allyl alcohol was compared in freshly isolated and cryopreserved hepatocytes that were either placed in suspension or maintained on hydrated collagen gels in a sandwich configuration. The purpose of this study was to evaluate whether the two types of cells displayed the same sensitivity to allyl alcohol when maintained in vitro over relatively prolonged periods of time. The important differentiated functions of urea synthesis, secretion of albumin, and metabolism of ethoxycoumarin, a model drug substrate, were used as end points of toxicity. Cryopreserved hepatocytes incubated in physiological buffer shortly after removal from liquid nitrogen were more sensitive to allyl alcohol than freshly isolated hepatocytes. In contrast, cryopreserved and freshly isolated hepatocytes maintained on hydrated collagen gels responded identically to allyl alcohol. Thus, the increased sensitivity of cryopreserved hepatocytes in suspension to allyl alcohol is a transient phenomenon that disappears after the cells have been allowed to recover on hydrated collagen gels. Dissipation of the mitochondrial membrane potential by allyl alcohol, as indexed by rhodamine 123 fluorescence, was also the same in freshly isolated and cryopreserved hepatocytes maintained on hydrated collagen matrices. This loss of mitochondrial membrane potential caused by allyl alcohol preceded inhibition of albumin and urea biosynthesis. Collectively, the results indicate that cryopreserved cells maintained on hydrated collagen gels provide a useful system to define the actions of certain hepatotoxic agents over relatively prolonged periods of time in vitro.