The deuterium washout technique of measuring tissue blood flow is based upon NMR detection of HOD (administered as D2O saline, but typically detected as HOD because of rapid proton‐deuteron exchange) as a freely diffusible tracer. Though this method is coming into more general use, it has not yet been rigorously validated in vivo against an accepted, independent measure of tissue blood flow. To this end, simultaneous radiolabeled micro‐sphere and HOD washout blood flow measurements were made in rat gastrocnemius muscle. D2O saline was administered either intramuscularly or intraarferially (near the aortic bifurcation), and the sciatic nerve was electrically stimulated to increase the muscle blood flow rate. Over a range of flows of 2 to 80 ml/(100 g. min), comparison of micro‐sphere and HOD washout measurements showed good agreement, with r = 0.92 (n = 16) for intramuscular administration and r = 0.91 (n = 12) for intraarterial administration. These data strongly suggest thatthe HOD washout technique provides accurate blood flow measurements in skeletal muscle.