The relationship between membrane potential and excitability for C-fibres has not been established, and repetitive activation of C-fibres gives rise to a slowing of impulse conduction velocity where the likely mechanism, previously ascribed to the electrogenic activity of the Na-pump, has been questioned recently by De Col et al. (2008). In order to shed light on these issues, we have explored the effects of changing membrane potential and also repetitive activation on the excitability and action currents recorded in rat primary cultured dorsal root ganglion (DRG) neurones (≤ 25μm in apparent diameter), in vitro. DRG neurones were maintained in culture for 24-48 hours and live stained with fluorescein conjugated IB4-lectin, allowing identification of IB4+ve and -ve populations. Current-clamp experiments were performed using whole-cell patch-clamp with quasiphysiological solutions and at 20-22°C. Neuronal membrane potential was initially held at -60 mV, incrementally hyperpolarized to near -100 mV, and then depolarized through -60 mV until the membrane ceased to be excitable. At all membrane potentials, threshold was measured in response to 10 ms current steps, applied at ≥ 2 second intervals. Over the potential range -100 to -20 mV, IB4 +ve neurones became more excitable with increasing depolarization until excitability was lost abruptly and completely positive to -20 mV. In contrast, IB4-ve neurones were most excitable near -55 mV (lowest threshold at: -26.3 ± 2.3 mV and -52.9 ± 6.0 mV, means ± S.E.M., IB4 +ve (n = 8) and IB4 -ve (n = 5), respectively; P< 0.01 Student’s t-test). We also report that repetitive, suprathreshold activation affected the amplitude of action currents. When holding the membrane potential of IB4 +ve neurones at -60 mV and stimulating at 2 Hz, inward transmembrane current was gradually suppressed during the train, and subsequently recovered at 0.5 Hz. Steady depolarization of the membrane potential to -50 mV caused a proportionally greater suppression of the inward current during a train, paralleling the effect of ouabain on C-fibre conduction latencies (De Col et al. 2008). IB4-ve neurones also showed suppression of inward transmembrane current, although the kinetics at -60 mV were more variable than for IB4 +ve. Our findings show the excitability of IB4 +ve neurones increases with membrane depolarization over a very wide membrane potential range, consistent with little adaptation, and also support the proposition that the entry of Na⁺ channels into inactivated states is a major factor in C-fibre conduction slowing during repetitive firing.