The influence of selective removal of the endocardial endothelium (by a 1-second exposure to the detergent Triton X-100, 0.5%) on myofilament Ca2+responsiveness and intracellular Ca2+transients was studied in ferret papillary muscles loaded with the Ca2+-regulated bioluminescent indicator aequorin. The removal of endocardial endothelium produced three major effects: 1) a decrease in peak developed tension and an early onset in isometric relaxation without corresponding changes in the intracellular Ca2+transient; 2) a rightward shift in the peak [Ca2+]i-peak tension relation with no change in maximum Ca2+-activated twitch tension; and 3) a decrease in steady-state tetanic force with a slight increase in the steady-state [Ca2+]i(at 4 mM [Ca2+]) and an unchanged steady-state tetanic force with a clear increase in the steady-state [Ca2+]i(at 10 mM [Ca2+]). These results suggest that intact endocardium may enhance performance of the heart by increasing the myofilament Ca2+responsiveness through endothelium-derived compounds such as endothelin. This hypothesis is supported by our observations that endothelin 1) induced a leftward shift in peak [Ca2+]i-peak tension curve and 2) could reverse the characteristic changes produced by the removal of endocardium.