When microspheres are injected into the circulation, they are trapped in the arteriolar or capillary system within various organs. It has been confirmed in animal experiments that the number of microspheres in a myocardial sample approximately follows a Poisson distribution, under adequate experimental conditions. On the basis of this result, we arrived at the following hypothesis: When regional blood flow is measured under a steady state by the reference sample method, the 95% relative error can be approximated by +/- 196 square root 1/v + 1/w, where v and w represent the number of microspheres in blood sample and myocardial sample, respectively. The equation is valid if v and w are greater than 400 and 49, respectively. We obtained an expression of the relation among the percent of increase or decrease in regional blood flow being verified, the probability of increase or decrease, as a statistically significant variation and the number of microspheres in a myocardial sample. An investigator can work out an approximate experimental design using this expression. For instance, when the increase in regional blood flow required for verification is expected to be 20% and he wants this increase to be verified by 90%-probability and as a statistically significant increase, he can predict from this expression that the number of microspheres in a myocardial sample should be 472 and 567 before and after the experimental intervention on the coronary circulation, respectively. The expression is useful as index for experiments involving use of the microsphere method.