It was the purpose of this study to examine the effect of a priming contractile bout on oxygen, uptake (V̇o2) on-kinetics in highly oxidative skeletal muscle. Canine gastrocnemii (n = 12) were stimulated via their sciatic nerves (8 V, 0.2-ms duration, 50 Hz, 200-ms train) at a rate of 2 contractions/3 s (≈70% peak V̇o2) for two 2-min bouts, separated by 2 min of recovery. Blood flow was recorded with an ultrasonic flowmeter, and muscle oxygenation monitored via near-infrared spectroscopy. Compared with the first bout (bout 2 vs. bout 1), the V̇o2 primary time constant (mean ± SD, 9.4 ± 2.3 vs. 12.0 ± 3.9 s) and slowcomponent amplitude (5.9 ± 6.3 vs. 12.1 ± 9.0 ml O 2˙kg wet wt-1˙min-1) were significantly reduced (P < 0.05) during the second bout. Blood flow on-kinetics were significantly speeded during the second bout (time constant = 7.7 ± 2.6 vs. 14.8 ± 5.8 s), and O2 extraction was greater at the onset of contractions (0.050 ± 0.030 vs. 0.020 ± 0.010 ml 02/ml blood). Kinetics of muscle deoxygenation were significantly slower at the onset of the second bout (7.2 ± 2.2 vs. 4.4 ± 1.2 s), while relative oxyhemoglobin concentration was elevated throughout the second bout. These results suggest that better matching of O2 delivery to V̇o2 speeds V̇o2 on-kinetics at this metabolic rate, but do not eliminate a potential role for enhanced metabolic activation. Additionally, altered motor unit recruitment at the onset of a second bout is not a prerequisite for reductions in the V̇o2 slow-component amplitude after a priming contractile bout in canine muscle in situ. Copyright © 2010 the American. Physiological Society.