We have examined the events underlying the initiation of spontaneous action potentials (fibrillation) in fibres of previously denervated rat diaphragm maintained in organ culture for up to 10 days.1. Based on discharge pattern, two classes of spontaneously active fibres were found: rhythmically discharging fibres, and fibres in which action potentials occur at irregular intervals.2. Sites of action potentials initiation were located by exploration along the fibre length with two independent extracellular recording electrodes. The majority of sites of origin in both regular and irregular fibres were at the former end-plate zone; however, there was no region along the length that could not, at least in some fibres, be a site of origin.3. Intracellular recording at or near sites of origin of action potential discharge showed two types of initiating events. Irregularly discharging fibres were brought to threshold by discrete depolarizations of up to 15 mV in amplitude, while regularly occurring action potentials were associated with oscillations of the membrane potential.4. Discrete depolarizations (called fibrillatory origin potentials or f.o.p.s) at sites of origin in irregularly discharging fibres have the following properties: (a) random occurrence and nearly constant amplitude outside a refractory period during which both amplitude and probability of a second f.o.p. are reduced; (b) associated inward current flow which is localized to about 100 mum or less along the fibre length, and (c) dependence of amplitude and frequency on membrane potential.5. Oscillation of membrane potential found at sites of origin of action potential discharge in regular fibres also occurred locally along the fibre length and was sensitive to changes in membrane potential.6. Both f.o.p.s and oscillations of membrane potential were reversibly abolished by low Na(+)-Ringer fluid or tetrodotoxin.7. Neither type of initiating event was appreciably affected by concentrations of D-tubocurarine which blocked extrajunctional sensitivity to acetylcholine.8. We conclude that spontaneous action potentials under these conditions arise from a localized Na(+)-conductance change in the membrane of the active fibre; this conductance change is distinct from the increased Na(+)-conductance which follows the interaction of acetylcholine with its receptor. Spontaneous activity in single, denervated muscle fibres is cyclical and self-inhibiting (Purves & Sakmann, 1974); thus the Na(+)-conductance change underlying the initiation of spontaneous action potentials is affected by muscle fibre activity.