We describe techniques for the isolation of Ca(2+)-tolerant myocytes from mouse (2 to 6 mo old) ventricle for measurements of mechanics of contraction and microfluorimetry. Our approach involved special modifications of existing methods that had been developed for other species but were not successful when applied to the mouse heart. Important features of the method are 1) a requirement for careful timing (< 5 min) of perfusion with nominally Ca(2+)-free solution; 2) perfusion with a solution containing a specially selected batch of collagenase in the presence of a low Ca2+ concentration; and 3) meticulous attention to water quality. Using this method, we could consistently isolate durable, Ca(2+)-tolerant myocytes from adult mouse hearts with a yield of approximately 50%. With slight modifications, the method should enable other investigators to isolate mouse cardiomyocytes for their specific experimental applications.