1. ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3). 2. Northern and western blot analyses demonstrated that mRNA and protein levels of the short isoform (sClC-3) in the heart were significantly increased in hsClC-3-overexpressing (OE) mice compared with wild-type (WT) mice. Heart weight : bodyweight ratios for OE mice were significantly smaller compared with age-matched WT mice. 3. Electrocardiogram recordings indicated no difference at rest, whereas echocardiographic recordings revealed consistent reductions in left ventricular diastolic diameter, left ventricular posterior wall thickness at end of diastole and interventricular septum thickness in diastole in OE mice. 4. The VSOAC current densities in atrial cardiomyocytes were significantly increased by ClC-3 overexpression compared with WT cells. No differences in VSOAC current properties in OE and WT atrial myocytes were observed in terms of outward rectification, anion permeability (I(-) > Cl(-) > Asp(-)) and inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and glibenclamide. The VSOAC in atrial myocytes from both groups were totally abolished by phorbol-12,13-dibutyrate (a protein kinase C activator) and by intracellular dialysis of an N-terminal anti-ClC-3 antibody. 5. Cardiac cell volume measurements revealed a significant acceleration of the rate of regulatory volume decrease (RVD) in OE myocytes compared with WT. 6. In conclusion, enhanced VSOAC currents and acceleration of the time-course of RVD in atrial myocytes of OE mice is strong evidence supporting an essential role of sClC-3 in native VSOAC function in mouse atrial myocytes.