Validation of the reference sample method (RSM) in the rat was accomplished by: 1) simultaneous measurement of cardiac output (CO) by the RSM and electromagnetic flowmetry method in ether-anesthetized open-chest Wistar rats, and 2) determination of systemic and regional hemodynamic reproducibility in conscious rats following three serial injections of microspheres. The optimal sampling time and sampling rate were determined in several CO states. The results indicate that: 1) 200 or more microspheres contained in the reference sample result in an accurate determination of CO, 2) almost 100% of the microspheres are trapped by tissues with in 20 s of cessation of the microsphere injection, 3) the hemodynamic steady state is affected only under low cardiac output conditions at a high sampling rate (1.211 ml/min), 4) three repeated injections of microspheres in conscious animals do not alter systemic hemodynamis, and 5) the reproducibility of regional blood flow distribution is good in all organs except the liver, stomach, spleen, and skin (third application). These data indicate that the RSM is reliable, providing reasonable accuracy and reproducibility of systemic and regional hemodynamics in the rat.