Cytochemical studies suggest that Na+,K+-ATPase is localized to sinusoidal and lateral portions of the hepatocyte plasma membrane whereas Mg++-ATPase and alkaline phosphatase are luminal or canalicular membrane markers. To validate further these cytochemical findings, we have isolated from the nuclear pellet of rat liver homogenates a liver plasma membrane (LPM) fraction enriched in all three enzyme markers, as previously described (Biochimica et Biophysica Acta 1975; 401:59-52). Following tight Dounce homogenization, the vesiculated membrane preparation was further separated on a multiple-step discontinuous sucrose density gradient (d 1.12 to 1.22). Na+,K+-ATPase activity "dissociated" from Mg++-ATPase activity, sedimenting at densities of 1.14 and greater. Further studies were carried out in two-step discontinuous sucrose gradients (1.13 and d greater than 1.13), and a light density fraction (d 1.13) was further characterized in calcium-free media (since addition of calcium increased contamination with intracellular membranes). Electron microscopy demonstrated a homogeneous vesicular membrane population in contrast to the heavy density fraction (d greater than 1.13) which contained membrane sheets and junction complexes as well as vesicles. The light density fraction was highly enriched n Mg++-ATPase (42.1 x homogenate specific activity) and alkaline phosphatase (64.6 x homogenate), 3 to 4 times their activities in the original LPM. In contrast, Na+,K+-ATPase activity in the light density fraction, diminished 16-fold from values in the original unfractionated LPM. All but 15% of total Na+,K+-ATPase activity in the original LPM could be accounted for in unwashed preparations. Neither cholesterol/phospholipid ratios nor an analysis of peptides on sodium dodecyl sulfate gel electrophoresis demonstrated differences in the composition of the light vs. heavy density subfractions, although there were relative increases in several peptide bands in the light density subfraction. These studies provide further supporting biochemical evidence for the concept that Na+,K+-ATPase resides on different membrane domains than does Mg++-ATPase and alkaline phosphatase and further characterizes a vesiculated membrane preparation highly enriched in putative "canalicular" enzyme markers.