Quantification of regional myocardial blood flow (MBF) with rubidium-82 (82Rb) and positron emission tomography (PET) requires the validation of a tracer kinetic model that adequately describes the kinetics of 82Rb in tissue. We performed 134 82Rb kinetic experiments in 24 isolated, arterially perfused rabbit interventricular septa at different flow rates (0.5-4.5 ml.min-1.g-1) and under the following conditions: varied concentrations of glucose (2.8-11 mM), insulin (0-5 mU/ml), and potassium (5-8 mM); varied pH (7.2-7.8); varied workload; and the addition of acetylstrophanthidin (1.0 microM). The measured 82Rb tissue time-activity curves consisted of two exponential components, with half times inversely related to MBF. The size of the slow component decreased as MBF increased (from 70% at 0.3 ml.min-1.g-1 to 30% at 2.0 ml. min-1.g-1). The kinetics were not strongly dependent on the conditions studied and were consistent with a compartmental model containing two communicating compartments. The volume of the fast exchangeable compartment and the transport rate constant from the fast to the slow compartment were found to be independent of MBF. Values of MBF were estimated with the compartmental model from 82Rb kinetics, and they correlated well with the directly measured MBF (r = 0.91; slope = 1.03).