Improved viability and function of insulin-producing beta (B) cells of frozen-stored human fetal pancreatic tissue was obtained by a two-step method utilizing high concentrations of dimethyl sulfoxide (DMSO). Human fetal pancreata (14–23–week gestation) obtained from pathologic abortions were teased and cultured overnight. Prior to freezing the tissues were immersed in 0.9% saline containing 0.5 M DMSO for 30 min (room temperature) and then placed in 2.1M DMSO on ice for 5 min. The tissues were frozen by the method previously developed in our laboratory and stored at – 196°C. The frozen-stored tissues were subsequently thawed at 24°C and cultured overnight before viability testing. Viability and function of the B cells were assessed by several specific assay methods; glucose plus theophylline-induced insulin release during static incubation and perifusion,3H-leucine incorporation into insulin, and insulin content of the tissue grown in athymic mice for 7 days.The response to glucose plus theophylline stimulation, measured on the frozen-thawed tissue one day after thawing, was 80% of the level measured in control tissue maintained in organ culture. Frozen-thawed tissues maintained in organ culture for 1 week responded comparably in the in vitro assay systems. The insulin content of frozen-thawed pancreatic tissue removed from athymic mice 1 week after transplantation was approximately 60% of the amount measured in the control grafts. These results demonstrate the utility of our procedure in the maintenance of the viability and function of frozen-stored human B cells both in culture and after transplantation.