This study investigates the relationship between the nutritional state of rats and lipid metabolism in distinct hepatic intracellular sites. Hepatic uptake of both protein and triacylglycerol (TG) moieties of injected very low-density lipoprotein (VLDL) is increased in fasted rats compared with fed controls. The VLDL-TG hydrolysis rate is increased in the plasma of fasted rats. This is shown by a higher ratio of labeled free fatty acid (FFA) to TG (FFA/TG). In both fed and fasted rats, a much greater increase of the labeled FFA/TG ratio in endosomes, compared with that in plasma, shows that further TG hydrolysis occurs in prelysosomal compartments. However, in fasted rats, this increase (18-fold) is much less than that in fed rats (69-fold). This observation is supported by the finding of significantly lower TG-lipase activity at pH 5, 7, and 8.6 in the endosomes of fasted rats. In contrast, during fasting, TG-lipase activity in whole liver homogenate and in isolated lysosomes is increased at pH 5. These observations suggest that after feeding there is a shift in intracellular lipolytic activity from lysosomes to prelysosomal organelles.