We continuously observed successive pairs of R-R intervals during atrial fibrillation and hypothesized that the shortest R-R interval for a given preceding R-R interval in a pair represents the functional refractory period of the atrioventricular node at that preceding interval. To test this hypothesis we simulated atrial fibrillation in 28 isolated cross-perfused canine hearts and obtained an R-R interval scatterplot by plotting the R-R intervals as a function of the immediately preceding R-R interval. This scatterplot enabled us to detect a series of the shortest R-R intervals for a wide range of preceding R-R intervals, and this allowed us to estimate the rate-dependent functional refractory period of the atrioventricular node in simulated atrial fibrillation. The estimated functional refractory periods correlated well with those measured by the conventional method (r = 0.93). We conclude that the proposed method makes it possible to estimate the rate-dependent functional refractory periods of the atrioventricular node in atrial fibrillation.