1. The influence of the interstimulus interval on twitch duration was analysed in isolated heart muscle of the rat. When the muscle was in the steady state at interstimulus intervals at 5 s a test interval was interposed and varied. Duration of twitch and action potential, sarcomere length and peak force of the test beats were measured. 2. Twitch force and duration increased when the test interval was increased from 0.4 to 10 s. This effect was abolished by inhibitors of sarcoplasmic reticulum function (ryanodine, caffeine, Sr2+). Hence, the interval dependence is controlled by the sarcoplasmic reticulum. 3. Post-extrasystolic potentiation, variation of [Ca2+]o and [Na+]o and blocking of iCa with nifedipine and Mn2+ led to large variations in force, reflecting variations in the amount of Ca2+ released from the sarcoplasmic reticulum. The effect on twitch duration was small, indicating that twitch duration was rather insensitive to the amount of released Ca2+, and not controlled by iCa and Na(+)-Ca2+ exchange. 4. Action potential duration was much shorter than twitch duration and, depending on the intervention, changes were in the same or in opposite direction. Hence, the action potential did not determine twitch duration. 5. Small variations in sarcomere length amongst test contractions were observed, but these variations could not account for the effects of the test interval. 6. It is proposed that the Ca2+ pump in the sarcoplasmic reticulum is activated during each contraction and inactivates slowly. Thus, after a short interval the pump is still activated and rapidly sequesters much of the released Ca2+ leading to a small twitch and rapid relaxation. This mechanism ensures proper relaxation and diastolic filling of the ventricle. The biochemical basis and implications of the hypothesis are discussed.