Cryopreservation is an effective method of islet storage and may facilitate clinical trials of islet transplantation. It was the aim of the present study to evaluate the in vitro viability of cryopreserved rat islets, including the response to nonglucose secretagogues and glucose oxidation. After pancreatic digestion via intraductal injection of collagenase, 75- to 200-micron Wistar rat islets were handpicked and cultured in RPMI 1640 (glucose 11.1 mmol/L) and randomized into two groups: control (cultured 20 to 24 hours at 37 degrees C) and cryopreserved (after 20 to 24 hours of culture at 37 degrees C, islets were cryopreserved according to Rajotte's protocol: freezing velocity, -0.25 degree C/min; thawing velocity, 200 degrees C/min). In the two groups, we evaluated recovery, insulin content per islet, staining viability (ethidium bromide/orange acridine; semiquantitative scoring, measuring the viable area of the islet from 0 = less viable to 3 = more viable), insulin secretion after glucose and nonglucose secretagogues, and oxidation of D-[U-14C]glucose. The results for the control group were always higher for the following: recovery (95.4% +/- 1.2% v 83.0% +/- 2.1%, P = .00), insulin content (2,203.9 +/- 335.2 v 1,443.3 +/- 171.8 microU/islet, P = .03), insulin secretion after 5.5 mmol/L glucose (61.3 +/- 8.0 v 28.3 +/- 3.4 microU/islet/90 min, p = .00), 16.7 mmol/L glucose (151.4 +/- 16.1 v 98.7 +/- 14.1 microU/islet/90 min, p = .03), 10 mmol/L L-leucine +10 mmol/L L-glutamine (125.6 +/- 27.9 v 56.8 +/- 6.4 microU/islet/90 min, P = .05), and 10 mmol/L L-arginine (202.5 +/- 27.5 v 128.8 +/- 14.2 microU/islet/90 min, P = .01), and glucose oxidation at 5.5 mmol/L (12.5 +/- 1.1 v 7.9 +/- 0.6 pmol/islet/120 min, P = .00) and at 16.7 mmol/L (26.1 +/- 2.6 v 14.3 +/- 1.6 pmol/islet/120 min, P = .00). No significant differences in staining viability were found between groups (2.35 and 2.48, respectively, P = .55). However, cryopreserved and control islets showed a significant increase in insulin secretion and glucose oxidation after increasing the glucose concentration from 5.5 to 16.7 mmol/L. We conclude that when glucose is increased, cryopreserved islets keep the capacity to increase insulin secretion, but cryopreservation produces a significant decrease in several islet viability characteristics. This decrease may be due to a decline of beta-cell number per islet and/or a decrease in the content of insulin per beta cell.