Kinetics of intracellular ice formation (IIF) for isolated rat hepatocytes was studied using a cryomicroscopy system. The effect of the cooling rate on IIF was investigated between 20 and 400 °C/min in isotonic solution. At 50 °C/min and below, none of the hepatocytes underwent IIF; whereas at 150 °C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF (50TIIF) was almost constant with an average value of -7.7 °C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. 50TIIF from isothermal temperature experiments was ~ -5 °C as opposed to -7.7 °C for constant cooling rate experiments. These experiments clearly demonstrated both the time and temperature dependence of IIF. On the other hand, in cooling experiments in the absence of extracellular ice, IIF was not observed until ~ -20 °C (at which temperature the whole suspension was frozen spontaneously) suggesting the involvement of the external ice in the initiation of IIF. The effect of dimethyl sulfoxide (Me2SO) on IIF was also quantified. 50TIIF decreased from -7.7 °C in the absence of Me2SO to -16.8 °C in 2.0 M Me2SO for a cooling rate of 400 °C/min. However, the cooling rate (between 75 and 400 °C/min) did not significantly affect 50TIIF (-8.7 °C) in 0.5 M Me2SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes.