1. The latency relaxation has been examined in single fibres from frog striated muscle with particular attention given to its possible relation to Ca(2+) release during excitation-contraction coupling.2. Latency relaxations were recorded at 19-23 degrees C from massively stimulated (0.2 msec pulses) single fibres using two selected RCA 5734 transducer tubes in a bridge circuit.3. The depth of the latency relaxation has its full value when stimulus strength is between 40 and 400% above twitch threshold. Stronger stimuli reversibly diminish the latency relaxation.4. The variation in depth of latency relaxation with sarcomere length was found similar to that reported previously for multifibre preparations but in single fibres the peak of the curve consists of a plateau between sarcomere lengths of 2.8 mu and 3.2 mu.5. Sucrose hypertonicity increases the depth of the latency relaxation at sarcomere lengths below 2.8 mu but above this length it has either no effect or a depressant effect depending on the degree of hypertonicity.6. The maximal depth of the latency relaxation (measured at 3 mu) averaged 0.23% of the maximal tetanus tension (measured at 2.2 mu) and was strongly correlated (r = 0.87) with the latter in forty-five single fibres.7. The maximal depth of the latency relaxation is not correlated with the number of sarcomeres in series in a fibre.8. The results of this study are shown to fully support and extend Sandow's (1966) hypothesis that the latency relaxation is caused by release of activator Ca(2+) from the sarcoplasmic reticulum.