Various parameters, including the nature and proportion of the constituents of the cryoprotective medium, the cooling rate, and the composition of the thawing medium, were evaluated for the cryopreservation of adult rat hepatocytes. The highest percentage of cells able to survive in culture was obtained by freezing in L15 medium containing 16% dimethyl sulfoxide, at a rate of 3 degrees C/min, and by adding 0.8 M glucose to the thawing medium. More than 50% of hepatocytes capable of attachment just after cell isolation kept this property after freezing and survived in primary culture. Dead cells could be eliminated before seeding by centrifugation on a Percoll layer. In culture, frozen cells exhibited a morphology similar to that of unfrozen cells and after 24 hr their protein secretion rate was reduced by only about 40%.