In the present study a technique was developed to demonstrate 5'-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4 degrees C rather than at 37 degrees C. The optimized incubation medium contained 300 mM Tris-maleate buffer, pH 7.2, 5 mM adenosine monophosphate as substrate, 30 mM cerium chloride as capturing agent for liberated phosphate, 10 mM magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5'-nucleotidase activity were obtained when incubations were performed at 37 degrees C and 4 degrees C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37 degrees C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8 micrograms. 5'-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37 degrees C than at 4 degrees C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37 degrees C, in comparison with that at 4 degrees C, were the appearance of multi-lamellar structures at bile canaliculi at 37 degrees C. We conclude that the present method is valid for the demonstration of 5'-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.