Duchenne muscular dystrophy (DMD), induced by mutations in the gene encoding for the intracellular protein dystrophin, is a severe inherited disease characterized by progressive muscle weakness and degeneration. Besides the well-studied skeletal muscle degenerative processes, DMD is also associated with cardiac complications. These include dilated cardiomyopathy development and cardiac arrhythmias. The present understanding of the pathomechanisms in the DMD heart is very limited, but evidence has been accumulating that dysfunctional ion channels in dystrophic cardiomyocytes play a significant role. The aim of this study was to characterize potential abnormalities in L-type calcium channel function, expression, and regulation in ventricular cardiomyocytes derived from dystrophic mdx mice. The mdx mouse is dystrophin-deficient, and the most commonly used mouse model for human DMD. For the experiments, ventricular cardiomyocytes were isolated from the hearts of adult normal (wild type) and dystrophic mdx mice, and the properties of their calcium channels were compared by using the whole cell patch-clamp technique. In addition, RT-PCR, western blot and immunocytochemical studies were performed. We found that currents through L-type calcium channels were significantly increased, and channel inactivation was reduced in dystrophic ventricular cardiomyocytes. These gain-of-function calcium current abnormalities will increase the Ca influx into the dystrophic myocyte during an action potential. Calcium channel subunit expression and localization in dystrophic cardiomyocytes was normal. This suggested that altered channel regulation was responsible for the observed calcium current abnormalities in dystrophic cells. Accordingly, inhibition of neuronal nitric oxide synthase (nNOS) increased the calcium currents only in wild type but not in dystrophic cardiomyocytes. This points to impaired nNOS activity in dystrophic cardiomyocytes as potential source for functional L-type calcium channel abnormalities. We conclude that gain-of-function L-type calcium channel abnormalities enhance the risk of calcium-dependent arrhythmias and cellular calcium overload in the dystrophic heart.