1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned alpha-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-S-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop. 3. SNS and SNS(SA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels. 4. Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I-II loop.