Unmyelinated nociceptive fibres are a key element in the human nociceptive system, however, it is very difficult to investigate such fibres in vivo in more detail. An alternate approach are studies on isolated human nerves. Here we describe that confocal Ca2+ imaging reveals new information about the physiology of human nociceptive C fibres. Confocal images at two emission wavelengths were collected from regions with unmyelinated nerve fibres within segments of biopsied human sural nerves stained with the Ca2+-sensitive fluorescent dyes Calcium Green-1 and Fura Red. Short trains of supramaximal electrical stimuli applied to one end of the nerve as well as bath application of capsaicin resulted in an increase in the free intracellular Ca2+ concentration. Intracellular Ca2+ transients were seen at action potential frequencies above 1 Hz. They were absent in Ca2+-free bathing solution and reduced during bath application of cadmium. This indicates an extracellular source of the activity-dependent rise in [Ca2+]i. Furthermore, Ca2+ transients were also observed during elevation of the extracellular K+ concentration or during short trains of calcium action potentials. Such 'Ca2+ spikes' were elicited by a combination of tetrodotoxin and potassium channel blockers. These data suggest the presence of voltage-dependent Ca2+ channels in the membrane of nociceptive human nerve fibres.