In athletic animals the spleen induces acute polycythemia by dynamic contraction that releases red blood cells into the circulation in response to increased O₂ demand and metabolic stress; when energy demand is relieved the polycythemia is rapidly reversed by splenic relaxation. We have shown in adult foxhounds that splenectomy eliminates exercise-induced polycythemia, thereby reducing peak O₂ uptake and lung diffusing capacity for carbon monoxide (DL_CO) as well as exaggerating pre-existing DL_CO impairment imposed by pneumonectomy (Dane et al., J. Appl. Physiol. 101:289-97, 2006). To examine whether the post-splenectomy reduction in DL_CO leads to abnormalities in O₂diffusion, ventilation-perfusion inequality or hemodynamic function, we studied these animals using the multiple inert gas elimination technique at rest and during exercise at a constant workload equivalent to 50% or 80% of peak O₂ uptake while breathing 21% and 14% O₂ in balanced order. From rest to exercise after splenectomy compared to before, minute ventilation was significantly elevated with respect to O₂ uptake; cardiac output, O₂ delivery and mean pulmonary and systemic arterial blood pressures were 10-20% lower while O₂ extraction was elevated with respect to O₂ uptake. Ventilation-perfusion inequality was unchanged, but O₂ diffusing capacities of lung (DL_O2) and peripheral tissue during exercise were lower with respect to cardiac output post-splenectomy by 32% and 25%, respectively. The relationship between DL_O2 and DL_CO was unchanged by splenectomy. We conclude that the canine spleen regulates both convective and diffusive O₂ transport during exercise to increase maximal O₂ uptake.