Interpretation of first-pass myocardial perfusion studies employing bolus administration of T1 magnetic resonance (MR) contrast agents requires an understanding of the relationship between contrast concentration and image pixel intensity. The potential effects of myocardial water exchange rates among the intravascular, interstitial, and cellular compartments on this relationship are controversial. We directly studied these issues in isolated, nonbeating canine interventricular septa. Myocardial T1 was measured three times/s during bolus transit of intravascular (albumin-Gd-DTPA and polylysine-Gd-DTPA) and extracellular (gadoteridol) contrast agents. For polylysine-Gd-DTPA, the peak changes in myocardial 1/T1 (delta R1) scaled nonlinearly with perfusate contrast concentration whereas a linear relationship would be expected for fast water exchange among the vascular, interstitial, and cellular compartments. For all agents, the peak delta R1 were much smaller than the values expected on the basis of fast myocardial water exchange. The data demonstrate that in isolated myocardial tissue, myocardial T1 enhancement during bolus administration of contrast can be strongly affected by myocardial water exchange for both intravascular and extracellular MR contrast agents.