Objective: Osmotic gradient-induced volume change and sarcolemmal water permeability of cardiac myocytes were evaluated to characterize the mechanism of water flux across the plasma membranes. Methods: Cell surface dimensions were measured from isolated guinea-pig and rat ventricular myocytes by digital videomicroscopy, and membrane hydraulic conductivity (L p ) was obtained by analyzing the time course of cell swelling and shrinkage in response to osmotic gradients. Results: Superfusion with anisosmotic solution (0.5-4 times normal osmolality) caused a rapid (<3 min to steady states) and reversible myocyte swelling or shrinkage. L p was ~1.9x10 - 1 0 l N - 1 s - 1 for guinea-pig myocytes and ~1.7x10 - 1 0 l N - 1 s - 1 for rat myocytes at 35 o C. Arrhenius activation energy (E a ), a measure of the energy barrier to water flux, was ~3.7 (guinea-pig) and ~3.6 kcal mol - 1 (rat) between 11 and 35 o C; these values are equivalent to E a of self-diffusion of water in bulk solution (~4 kcal mol - 1 ). Treatment with 0.1 mM Hg 2 + , a sulfhydryl-oxidizing reagent that blocks membrane water channels, reduced L p by ~80%, and the sulfhydryl-reducing reagent dithiothreitol (10 mM) antagonized the inhibitory action of Hg 2 + . Inhibition of the volume-sensitive cation (30 μM Gd 3 + ) and anion (1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate) channels and Na + -K + pump (10 μM ouabain) modified the size of osmotic swelling but had little effect on L p . Conclusions: Although the observed L p is relatively small in magnitude, the low E a and the sulfhydryl reagent-induced modification of L p are characteristic of channel-mediated water transport. These data suggest that water flux across the sarcolemma of guinea-pig and rat heart cells occurs through parallel pathways, i.e., the majority passing through water channels and the remainder penetrating the lipid bilayers.