We have shown previously that rat sciatic nerve axons in vitro express sensitivity to capsaicin and heat and responded to these stimuli with a Ca2+-dependent and graded immunoreactive calcitonin gene-related peptide release. Morphological evidence for stimulated vesicular exocytosis and for the vanilloid receptor TRPV1 in the axolemma of the unmyelinated nerve fibres has also been presented. Here we used solutions of low pH, high K+ or 47 degrees C to stimulate isolated desheathed sciatic nerves measuring immunoreactive calcitonin gene-related peptide release. pH 6.1 increased immunoreactive calcitonin gene-related peptide release by 31% over baseline and pH 5.2 and 4.3 caused a log-linear concentration-dependent increase of 137 and 265%, respectively. The effect of pH 3.4 was out of the linear range and not reversible. Stimulation in Ca2+-free solutions and under increased intracellular Ca2+ buffering capacity strongly reduced the proton responses. The TRPV1 antagonists capsazepine and ruthenium red substantially reduced the effects of pH 5.2 but not pH 6.1. Combining a stimulus of 60 mm K+ with the subliminal pH 6.3 reduced the axonal immunoreactive calcitonin gene-related peptide response by 88%. The noxious heat response at pH 6.3, however, was only reduced by 39%, suggesting a hidden sensitization to heat by low pH. This was supported by an effect of capsazepine to reduce the combined response to half, indicative of an involvement of TRPV1 in the sensitization but not in the axonal heat response itself that was found to be resistant to capsazepine. Axonal calcitonin gene-related peptide release is thought to play a physiological role in activity-dependent autoregulation of endoneurial blood flow. Axonal sensitivity to and sensitization by protons may be a pathophysiological mechanism involved in certain peripheral neuropathies.