We have developed methodology to simultaneously measure fluid redistribution among the major compartments during moderate and severe hypohydration. Total body water (TBW) was determined using tritiated water, extracellular fluid volume (ECF) was measured using a single-injection [14C]inulin technique, and plasma volume (PV) was determined by indocyanine green dye dilution. Moderate (10% decrease in body wt) and severe (15%) hypohydration resulted in significant losses in TBW, ECF, and PV. Plasma volume was decreased by approximately 25% in both groups, and other fluid compartments were differentially affected. For example, the moderately dehydrated group maintained PV by shifting fluid from the interstitial fluid volume (ISF) compartment while preserving the intracellular fluid volume (ICF); conversely, the severely dehydrated group maintained PV by redistributing fluid from both the ISF and ICF compartments. The data indicated that the initial response to fluid loss was the movement of fluid from the ISF pool to sustain both PV and ICF. In severely hypohydrated rats, PV was maintained at the expense of ICF. These experiments indicated that PV and ICF were maximally protected, probably to preserve the integrity of the cardiovascular system and to minimize organ injury.