The relationship between different frequencies of loading stimulation and [3H]acetylcholine (ACh) formation and release from nerve terminals has been investigated in extratrigonal strips of the urinary bladder of the rat. An increase in frequency (0.2, 0.4 and 0.8 Hz) for the 30 min incubations with [3H]choline produced an enhancement of storage of [3H]ACh from 19.5 to 34% of total tritium content in the tissue. Higher frequencies (1.6 and 3.2 Hz) failed to increase storage further on. The [3H]choline content did not vary significantly. Electrical field stimulation at 2 Hz (360 shocks) produced a release of tritium. The evoked outflow was higher when the strip was loaded at 0.8 Hz than at the other frequencies tested. Both [3H]ACh and [3H]choline were measured in the perfusate of strips preloaded at 0.8 Hz. Most of the induced outflow was found to be [3H]ACh, as in previous experiments carried out using 0.2 Hz as a loading frequency. The findings suggest that in the rat urinary bladder strip loading at 0.8 Hz is suitable for increasing the formation and the resulting release of [3H]ACh during electrical stimulation.