The present study describes an experimental setup that enables continuous measurement of cellular volumes in isolated organs. The procedure is a modification of a recently reported method that uses multinuclear NMR measured by 59Co NMR of cobalticyanide and 1H NMR of water in isolated rat hearts at normothermia. The new apparatus contains a background flow which is shown to improve the rate of exchange of the marker between the interstitium and the external solution and allows detection of cellular shrinkage during no-flow ischemia. A series of experiments of marker loading and wash-out were performed to validate the method. In the Langendorff preparation, intracellular volumes (in units of milliliters per gram dry weight) of hearts perfused with Krebs-Henseleit solution oscillated around a mean value of 2.50 +/- 0.06 ml/gdw. During 30 min of ischemia the cells swelled to 2.88 +/- 0.08 ml/gdw and residual edema was observed after 30 min of reperfusion (2.62 +/- 0.08 ml/gdw). A hypoosmotic shock was used to assess changes in membrane permeability at different time points of ischemia and reperfusion. Water influx induced by the hypoosmotic shock at the end of ischemia was similar to that elicited in perfused hearts. After 15 and 30 min of reperfusion, the magnitude of the response to hypoosmolarity decreased by 9 and 37%, respectively, indicating a gradual permeabilization of the membranes, presumably to ions. The experimental setup was also used to monitor intracellular volumes as a function of time in anisoosmotic conditions. Cellular swelling/shrinkage were delayed for periods of 5 and 8 min at osmolarities of +/-50 and +/-100 mosmol/liter, suggesting a limited capability of the heart to absorb an anisoosmotic shock. The variation in cellular volumes was proportional to the deviation of the conditions from isoosmolarity, and activation of volume-regulatory mechanisms was demonstrated. The noninvasive technique presented in this study is capable of providing quantitative evidence of changes in cellular volumes in isolated hearts at a temporal resolution of 1 min and a spatial resolution of 4% (of cellular volume). As demonstrated in the cases of global ischemia and anisoosmolar conditions, the technique is expected to provide new insights into the mechanism of cellular-volume regulation.