Rat islet cells, dissociated with EDTA-Dispase, were immersed in 10% dimethyl sulfoxide at 20 degrees C for 15 min and frozen to -40 degrees C at a cooling rate of 0.5 or 1.0 degree C/min and subsequently further to -80 degrees C at 3 degrees C/min by a programmable freezer. After being maintained at -80 degrees C for 10 min, they were rapidly thawed in a water bath at 37 degrees C. They were cultured for 12 h and preincubated in 3.3 mM glucose-containing Krebs-Henseleit bicarbonate buffer (KHBB) for 1 h. Groups of 10(4) cells were then incubated in 3.3 or 16.7 mM glucose-containing KHBB for another hour. As a control, non-frozen-thawed cultured islet cells were incubated similarly. The non-frozen rat islet cells released 1.29 pg insulin/cell.60 min in the presence of 3.3 mM glucose and this release level was significantly elevated to 1.64 pg insulin/cell.60 min in the presence of 16.7 mM glucose. The cells frozen at 0.5 degree C/min releasing 1.55 pg insulin/cell.60 min in the presence of 3.3 mM glucose also responded to 16.7 mM glucose and released the significantly high level of 1.87 pg insulin/cell.60 min. However, the islet cells frozen at a cooling rate of 1 degree C/min secreted 1.74 and 1.92 pg insulin/cell.60 min in the presence of 3.3 mM and 16.7 mM glucose respectively. There was no significant difference between these levels. These results indicate that cryopreservation at a cooling rate of 0.5 degree C/min may be adequate for the preservation of dispersed pancreatic endocrine cells.