We have demonstrated that the fluorescent microsphere technique can be used in small mammals for accurate determination of regional blood flows. Hn particular we have shown that 100% recovery of trapped microspheres is possible, that tissue digestion can be completed in a shorter time than previously reported, and the error-prone filtration method can be replaced with one of sedimentation. The method gave very good agreement among different fluorescent labels (r(2) > 0.99) and low variability among tissues (mean coefficient of variation = 0.06). Simultaneous injection of radiolabelled and fluorescent microspheres established comparability between these methods (r(2) = 0.96) for blood flows measured at rest, during vasodilator-induced hypotension, and in muscle hyperaemia during indirect electrical stimulation. Fluorescent microspheres can therefore replace radioactive microspheres for the determination of blood flow with advantages in both safety and cost, without loss of sensitivity.