To investigate the effects of water exchange on quantification of perfusion, data were acquired in isolated hearts (n = 11) and used to develop a model of exchange. Myocardial T1 was measured 3 times/sec during step changes in concentration of intravascular (polylysine-gadolinium-diethylene-triamine-pentaacetic acid) and extracellular (gadoteridol) agents. For the intravascular agent, the change in 1/T1 (deltaR1) was lower than predicted by fast exchange (2.7+/-0.5 vs. 7.8 sec(-1), respectively), and suggested an intra-extravascular exchange rate of 3 Hz. For the extracellular agent, contrast kinetics were similar to those of similarly sized molecules (wash-in time constant 38+/-5 sec), and the data suggested fast interstitial-cellular exchange. Modeling showed that perfusion is underestimated for both agents if exchange is ignored, although the relationships of measured to actual perfusion were monotonic. We conclude that myocardial water exchange strongly affects first-pass enhancement but that ignoring the effects of exchange may still provide reasonable estimates of regional perfusion differences.