An intrinsic birefringence signal with two components occurring before sarcomere shortening was measured in mammalian cardiac muscle. The second component was sensitive to the inotropic state of the muscle as affected by external calcium concentration and epinephrine but not by changes of resting length. The second component was absent in frog heart. These results suggest that the second component of the birefringence signal reflects the activity of the sarcoplasmic reticulum related to excitation-contraction coupling processes occurring prior to onset of contraction in mammalian cardiac muscle.