Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage-gated sodium channel alpha-subunits. However, of the auxiliary subunits, beta1 is expressed preferentially in only large- and medium-diameter neurons of the DRG while beta2 is absent in all DRG cells. In view of this, we have compared the distribution of beta1 in rat DRG and spinal cord with a novel, recently cloned beta1-like subunit, beta3. In situ hybridization studies demonstrated high levels of beta3 mRNA in small-diameter c-fibres, while beta1 mRNA was virtually absent in these cell types but was expressed in 100% of large-diameter neurons. In the spinal cord, beta3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while beta1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of beta3 expression in DRG appears to correlate with the TTX-resistant voltage-gated sodium channel subunit PN3, we co-expressed the two subunits in Xenopus oocytes. In this system, beta3 caused a 5-mV hyperpolarizing shift in the threshold of activation of PN3, and a threefold increase in the peak current amplitude when compared with PN3 expressed alone. On the basis of these results, we examined the expression of beta-subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in beta3 mRNA expression in small-diameter sensory neurons of the ipsilateral DRG. These results show that beta3 is the dominant auxiliary sodium channel subunit in small-diameter neurons of the rat DRG and that it is significantly upregulated in a model of neuropathic pain.