Levels of total, unesterified and esterified retinol were determined in liver, liver parenchymal cells (PC) and liver nonparenchymal cells (NPC) during vitamin A depletion in rats. Liver vitamin A levels decreased from 113 to 4 micrograms over a 97-d experimental period; plasma retinol concentrations did not change significantly during this time. Initially, greater than 90% of hepatic vitamin A was in the esterified form and most (93%) was localized in NPC. During vitamin A depletion, there were significant declines in retinyl ester content of both PC and NPC, but unesterified retinol levels were not significantly affected. Plasma retinol concentrations were significantly correlated with unesterified retinol mass in PC and NPC, but not with retinyl ester mass. Although 94% of the liver's negative vitamin A balance was due to changes in NPC retinyl ester levels, the fractional rate of retinyl ester loss from PC and NPC was almost identical. Since unesterified retinol levels in plasma, PC and NPC appeared to be conserved even when liver retinyl ester stores were virtually depleted, and since the retinol utilization rate was apparently not decreasing during this stage of vitamin A depletion, these data support the hypotheses that homeostatic mechanisms controlling the three pools of unesterified retinol are linked, and that vitamin A utilization rate is maintained as long as unesterified retinol levels in plasma, PC and NPC are normal.