Hemodilution reduces blood viscosity and O2 content (CaO2) and increases cerebral blood flow (CBF). Viscosity and CaO2 may contribute to increasing CBF after hemodilution. However, because hematocrit is the major contributor to blood viscosity and CaO2, it has been difficult to assess their relative importance. By varying blood viscosity without changing CaO2, prior investigation in hemodiluted animals has suggested that both factors play roughly equal roles. To further investigate the relationship of hemodilution, blood viscosity, CaO2, and CBF, we took the opposite approach in hemodiluted animals, i.e., we varied CaO2 without changing blood viscosity. Hyperbaric O2 was used to restore CaO2 to normal after hemodilution. Pentobarbital sodium-anesthetized rats underwent isovolumic hemodilution with 6% hetastarch, and forebrain CBF was measured with [3H]nicotine. One group of animals did not undergo hemodilution and served as controls (Con). In the three experimental groups, hematocrit was reduced from 44% to 17-19%. Con and hemodiluted (HDil) groups were ventilated with 40% O2 at 101 kPa (1 atmosphere absolute), which resulted in CaO2 values of 19.7 +/- 1.3 and 8.1 +/- 0.7 (SD) ml O2/dl, respectively. A second group of hemodiluted animals (HBar) was ventilated with 100% O2 at 506 kPa (5 atmospheres absolute) in a hyperbaric chamber, which restored CaO2 to an estimated 18.5 +/- 0.5 ml O2/dl by increasing dissolved O2. A fourth group of hemodiluted animals (HCon) served as hyperbaric controls and were ventilated with 10% O2 at 506 kPa, resulting in CaO2 of 9.1 +/- 0.6 ml O2/dl. CBF was 79 +/- 19 ml. 100 g-1. min-1 in the Con group and significantly increased to 123 +/- 9 ml. 100 g-1. min-1 in the HDil group. When CaO2 was restored to baseline with dissolved O2 in the HBar group, CBF decreased to 104 +/- 20 ml. 100 g-1. min-1. When normoxia was maintained during hyperbaric exposure in the HCon group, CBF was 125 +/- 18 ml. 100 g-1. min-1, a value indistinguishable from that in normobaric HDil animals. Our data demonstrate that the reduction in CaO2 after hemodilution is responsible for 40-60% of the increase in CBF.