The effects of 2,5-di-tert-butylhydroquinone (TBQ) on cardiac muscle contractility were investigated using cardiac preparations from the right ventricle of the rat. In saponin-skinned preparations, TBQ (50 and 100 microM) did not significantly affect the maximal isometric Ca2+-activated force or the steady-state force-Ca2+ concentration relation but significantly reduced the Ca2+ content of the sarcoplasmic reticulum (SR) in both the absence and the presence of 50 microM of the SR Ca2+-release channel blocker ruthenium red. The effect of TBQ on SR Ca2+ content was not due to an increased passive Ca2+ leak or Ca2+-induced Ca2+ release from the SR but to the specific inhibition of the SR Ca2+ pump. In electrically stimulated (0.2 Hz, 23 degrees C) intact cardiac preparations loaded with the Ca2+-sensitive fluorescent dye fura 2, TBQ (50 and 100 microM) caused the decrease in the rates of rise and fall of the fura 2 signal (ratio of fluorescence intensities at 360- and 380-nm excitation) and isometric force. In the presence of 50 and 100 microM TBQ, the twitch height of the isometric force response was significantly decreased, whereas the peak of the fura 2 signal was not significantly affected. At 2-Hz stimulation frequency, both parameters were reduced in the presence of 50 and 100 microM TBQ. Taken together, the results obtained in this study show that TBQ inhibits rather specifically the SR Ca2+ uptake in the rat ventricle and that it can be used as a tool to determine the function of the SR under different conditions.