Suspensions of enzymatically prepared hepatocytes from starved rats were separated according to their buoyant density at 12 degrees C in linear, isosmotic gradients of metrizamide, centrofuged at low speed for a relatively short time. The recovery of cell protein was 86%. Hepatocytes of high viability formed a single band around 1.10 g/cm3 and were recovered as four density populations (P1-P4) form low to high density, respectively. The content of protein was significantly lower in population P1, while the content of neutral fat or the averaged cell size was similar in the various populations. The specific activity of alanine aminotransferase increased in the order P1-P4. The distribution of this enzyme within the intact liver acinus obtained by others indicate that a partial separation of periportal and perivenous hepatocytes had occurred. The activity patterns of lactate dehydrogenase, glutamate dehydrogenase, isocitrate dehydrogenase (NADP+) and pyruvate kinase, also with known intra acinar distributions, supported this conclusion. The hepatocytes showed signs of shrinkage after separation, but since they retained a normal ultrastructure, most enzyme activities and viability, the present technique was regarded superior to previous procedures of hepatocyte separation by density. The degree of separation was calculated from an equation (see Appendix), and the periportal/perivenous ratio for parameters measured in density populations can be obtained. The specific activity of phosphofructokinase, alcohol dehydrogenase and aldehyde dehydrogenase showed no differences between populations. However, the ratio high-Km/low-Km aldehyde dehydrogenase increased in the order P4-P1.