To determine the contribution of haemoglobin (Hb) to the hypoxia-tolerance of Daphnia magna, we exposed Hb-poor and Hb-rich individuals (2.4-2.8 mm long) to a stepwise decrease in ambient oxygen partial pressure (P(O(2)amb)) over a period of 51 min from normoxia (20.56 kPa) to anoxia (<0.27 kPa) and looked for differences in their physiological performance. The haem-based concentrations of Hb in the haemolymph were 49 micromol l(-1) in Hb-poor and 337 micromol l(-1) in Hb-rich animals, respectively. The experimental apparatus made simultaneous measurement of appendage beating rate (fA), NADH fluorescence intensity (I(NADH)) of the appendage muscles, heart rate (fH) and in vivo Hb oxygen-saturation possible. In response to progressive, moderate hypoxia, both groups showed pronounced tachycardia and a slight decrease in fA. The fA and fH of Hb-rich animals were generally 4-6 % lower than those of Hb-poor animals. In addition, Hb-rich animals showed a significant decrease in the P(O(2)amb) at which the Hb in the heart region was half-saturated and a striking reduction in the critical P(O(2)amb) of appendage-related variables. In Hb-poor animals, the I(NADH) signal indicated that the oxygen supply to the limb muscle tissue started to become impeded at a critical P(O(2)amb) of 4.75 kPa, although the high level of fA was largely maintained until 1.77 kPa. The obvious discrepancy between these two critical P(O(2)amb) values suggested an anaerobic supplementation of energy provision in the range 4.75-1.77 kPa. The fact that I(NADH) of Hb-rich animals did not rise until P(O(2)amb) fell below 1.32 kPa strongly suggests that the extra Hb available to Hb-rich animals ensured an adequate oxygen supply to the limb muscle tissue in the P(O(2)amb) range 4.75-1.32 kPa. This finding illustrates the physiological benefit of Hb in enabling the animal to sustain its aerobic metabolism as the energetically most efficient mode of fuel utilization under conditions of reduced oxygen availability.