Cryopreservation of hepatocytes is a crucial step in the implementation of cell therapy for treating certain liver diseases. In the present study we investigated the effect of the some disaccharides on the cryopreservation of rat hepatocytes. Liver cells were frozen in media in the presence or absence of low concentrations of Me2SO (5% Me2SO) supplemented with varying concentrations of disaccharides (sucrose, glucose and trehalose). After 7 days of cryopreservation, the hepatocytes were thawed and viability was measure by exclusion of trypan blue and by the MTT technique, as well as by determining albumin production. Among the investigated disaccharides and concentrations, 0.2 M trehalose showed the best overall outcome. Compared to the use of Me2SO alone, significant improvement in post-thaw cell viability was observed. The new solution may reduce Me2SO side effects on patients and improve the viability and quality of cryopreserved hepatocytes.