To gain insight into diaphragm functional heterogeneity, blood flow (expressed as ml.min-1 x 100 g-1) was measured using radiolabeled microspheres in the ventral, medial, and dorsal regions of the costal diaphragm and in the crural diaphragm of sedentary control (S) and exercise trained (ET) female Wistar-Kyoto rats at rest and during treadmill exercise. ET animals had performed moderate intensity exercise training on a motorized treadmill (22 m/min, 10% grade, 60 min/d) for 12 months, while S were cage-confined. The efficacy of exercise training was demonstrated by a 12% increase (P < 0.05) in ventricular weight-to-body weight ratio and increases (P < 0.05) in citrate synthase activity in hindlimb skeletal muscles of ET. At rest, blood flow in the ventral costal diaphragm (16 +/- 1) averaged approximately 61% of that in the medial (26 +/- 3) and dorsal (25 +/- 2) costal regions (P = 0.035), and crural diaphragm flow was 23 +/- 3. During treadmill exercise (5 min at 22 m/min, 10% incline), blood flow increased an average of 5-fold (P < 0.001) throughout the diaphragm, but the heterogeneous flow pattern persisted; i.e., blood flow remained lower (P = 0.003) in the ventral region (77 +/- 7) than either the medial (135 +/- 15) or dorsal (127 +/- 11) costal regions. Flow in the crural diaphragm during exercise was intermediate (105 +/- 9). Exercise training did not alter either the magnitude of blood flows or the flow distribution pattern within the diaphragm. Citrate synthase activity was two-fold that of the plantaris muscle and was uniform across the ventral, medial, and dorsal costal and the crural diaphragm of a second group of age-matched rats (P = 0.57). These data demonstrate that, although oxidative capacity is uniform throughout the diaphragm, there is a significant regional heterogeneity of blood flow within the rat diaphragm both at rest and during locomotory exercise. The greater flow in the medial and dorsal regions of the costal diaphragm suggests that these regions sustain a greater portion of the inspiratory work load at rest and during exercise compared to the ventral region.