Previous studies have shown that the right ventricle (RV) appears to tolerate volume overloading better than does the left ventricle (LV). To determine whether this dichotomous response is due to intrinsic differences in the contractile performance of volume hypertrophied RV vs. LV cardiac muscle cells, or cardiocytes, we characterized the contractile performance of cardiocytes isolated from an experimental feline model of biventricular volume overload (n = 7 cats), wherein a peripheral arteriovenous fistula (AVF) produced an identical duration and degree of volume overload for both ventricles; sham-operated cats served as the appropriate controls (n = 7). Cardiocyte contractile function was defined by laser diffraction measurement of sarcomere motion. For the AVF cats, there was a 2.4-fold increase in cardiac output, a significant increase in the RV and LV weight-to-body weight ratios, and a significant increase in cell surface area for the RV and LV cardiocytes. Despite this, cardiocyte contractile function in the AVF cats was entirely normal. Thus there was no significant difference between RV and LV cardiocytes from control cats in the extent or velocity of sarcomere shortening either in 1 cP superfusate or in higher viscosity superfusates, and comparison of RV and LV cells from the AVF cats to those from sham-operated cats again revealed no significant differences. Furthermore, cardiocyte relengthening properties showed no significant differences between AVF and control groups. In summary, this study shows that contractile dysfunction is not an inherent property of either RV or LV cardiac muscle cells hypertrophying in response to substantial volume overloading when preload is increased but afterload is normal.