To obtain sufficient quantities of human fetal pancreatic tissue (HFP) for transplantation, long-term storage of the tissue must be achieved. The functional viability of HFP after cryopreservation by stepwise addition of dimethyl sulfoxide was determined. Human fetal pancreas explants (1-2 mm3; gestational age 16-21 wk) were prepared and slowly cooled (0.25 degrees C/min) to -40 degrees C before placement in liquid nitrogen. After rapid thaw (37 degrees C) and overnight culture, insulin release in response to high glucose and theophylline challenge determined in vitro functional viability. No difference was found between the responses of fresh and cryopreserved tissue (fresh 29.2 +/- 3.0 ng/mg tissue, cryopreserved 29.9 +/- 3.3 ng/mg tissue; P greater than .1). Diabetic BALB/c nu/nu mice transplanted with cryopreserved HFP returned to normoglycemia in 11 +/- 2 wk (range 8-15 wk). Oral glucose tolerance tests indicated in vivo serum glucose control equivalent to or better than that of nondiabetic control mice (peak serum glucose of nondiabetic mice 164 +/- 66 mg/dl, of cryopreserved grafted mice 180 +/- 67 mg/dl; P greater than .8). In vitro insulin release of cryopreserved grafted tissue demonstrated that the tissue had differentiated and matured and was now capable of responding to high-glucose challenge (39.3 +/- 11.5 ng insulin released/mg tissue). The results described herein are the first demonstration that cryopreserved HFP maintains the in vivo capacity to differentiate and mature and the capacity to reverse diabetes in an animal system.