Partially purified liver plasma membranes were fractionated further on sucrose layers. Three membrane populations, numbered Peaks 1, 2 and 3, were isolated at densities of 1.23, 1.16, and 1.03, respectively. Peaks 1 and 2 were enriched to a similar degree in 5′-nucleotidase activity, a plasma membrane marker, relative to membranes in Peak 3. Electron micrographs indicated that Peak 1 possessed desmosomes and bile canaliculi, while Peak 2 contained large vesicles as well as smaller vesicular structures attached to membranes. The latter have been attributed to hepatocyte sinusoidal surfaces. All three membrane fractions contained adenylate cyclase activity with the highest specific activity found in Peak 2. The enzyme in all three peaks was F sensitive with higher sensitivity in Peaks 1 and 2. Glucagon sensitivity of adenylate cyclase in Peak 2 membranes was four times that of Peak 1. Only Peak 2 membranes were sensitive to epinephrine. The Peak 2 membranes were three times more sensitive to glucagon than the partially purified membranes from which they were derived. These findings indicate that, while both bile canalicular and sinusoidal faces of hepatocytes possess adenylate cyclase, the sinusoidal fraction is more sensitive to glucagon. Solubilized adenylate cyclase of the Peak 2 membranes, obtained as the 165,000 g supernate of membranes treated with Lubrol-PX, was sensitive to stimulation by guanyl nucleotide analogs. Guanyl nucleotide sensitivity thus resides in the catalytic site and is not dependent on membrane integrity. All three membrane fractions possessed similar activities of nucleotide phosphohydrolase activity.