1. Adult rat flexor digitorum brevis muscles were dissociated by treatment with collagenase and trituration. Several hundred isolated fibres were obtained from each muscle. 2. Most isolated fibres appeared to be intact as judged by some morphological and physiological criteria, although resting membrane potentials were about -60 mV, which is somewhat lower than normal. 3. A small percentage of the muscle fibres were branched. 4. Acetylcholine sensitivity was measured iontophoretically. The sensitivity fell abruptly outside the margin of the end-plate. Extrajunctional sensitivty was detected on all fibres, and declined smoothly away from the end-plate to an undetectable level over a distance of about 200 micron. On a few fibres, ACh sensitivity was mapped circumferentially from the end-plate. It appeared to decline with distance in a manner similar to the longitudinal sensitivity gradient. 5. Fibres dissociated from muscles denervated a week earlier were sensitive to ACh everywhere on their surfaces.