The aim of this study was to validate in rats an alternative to the radioactive microspheres (RM) reference technique, the fluorescent microspheres (FM), for the simultaneous determination of cardiac index (CI) and regional blood flows (RBF). Validation of the FM method was performed in three steps: (a) comparison of CI and RBF values obtained simultaneously by FM and RM, (b) determination of the repeatability of the measurements by using two successive injections of FM, and (c) evaluation of the ability of the FM method to assess vasodilating effects (by using dipyridamole). CI values (range, 242-513 ml/min x kg; n = 20) obtained with FM correlated with those obtained with RM (r = 0.82: p<0.001), and agreement was found between FM and RM (error 95% confidence interval for one pair, +/-125 ml/min x kg). FM RBF values, although smaller than corresponding RM RBF values, were correlated with the latter (range, 0.1-7 ml/min x g; n = 71; r = 0.99; p< 0.001). Agreement was dependent on RBF values, e.g., error 95% confidence intervals for one pair were 0.08-0.13 and 3.86-6.48 for 0.1 and 5 ml/min x g, respectively. Two successive FM injections at a 10-min interval (conscious rats) provided similar values (n = 14) for CI (306+/-24 vs. 346+/- 18 ml/min x kg), and renal (5.1+/-0.2 vs. 6.2+/-0.3 ml/min x g), left (6.1+/-0.3 vs. 5.8+/-0.4 ml/min x g) and right (4.8+/-0.4 vs. 4.7+/-0.3 ml/min x g) myocardial RBF. Corresponding error 95% confidence intervals were +/-187 ml/min x kg, +/-2.8, +/-2.2, and +/-2.0 ml/min x g, respectively. Dipyridamole (2, 4, 6, and 8 mg/kg x min for 10 min, i.v.; n = 9-13 per group, conscious rats) significantly and dose dependently increased left and right myocardial blood flows, whereas renal blood flow was not affected. We conclude that the FM technique (a) is reliable and in agreement with the RM method, (b) provides repeatable measurements of systemic and regional hemodynamics, and (c) allows detection and quantification of vasodilating effects in rats.