Comparisons of the distributions of tracers putatively confined to the myocardial extracellular space (ECS) in vivo and in isolated hearts perfused with crystalloid solutions via their coronaries, generally indicate that ECS markedly expands—both absolutely and relative to cellular space (ICS)—over a period of in vitro perfusion. Quantification of the expansion is problematic, however, because of the difficulty of distinguishing between a true increase in anatomical ECS and expansion of tracer space due to penetration of the plasma membrane. This difficulty in turn renders an assessment of the distribution of electrolytes and other substances unreliable. In the present study a combined morphometric-tracer distribution analysis is applied towards distinguishing between ECS expansion and tracer leakage in the rat Langendorff preparation perfused with a modified Krebs-Henseleit solution. The results demonstrate that both ICS and ECS expanded with the first few minutes of perfusion, with the ICS remaining constant thereafter and the ECS increment declining slowly. Although the expansion of the ECS initially is disproportionately greater relative to that of the ICS, this relative difference disappears in about 1 h, i.e., the uptake of water by the myocardium is nearly homogeneous. An apparent increase of ECS relative to ICS, by more than one-third the initial space after 90 min perfusion, was found to be entirely artifactual and apparently due to a non-specific increase in sarcolemmal permeability.