1. The role of presumed chemical mediators of inflammation in the heat-induced sensitization of cutaneous C-polymodal nociceptors (CPNs) was examined in a rabbit ear preparation maintained in vitro by intra-arterial perfusion with a solution free of protein and cellular elements. 2. In this preparation, CPNs consistently showed enhanced responsiveness after repeated exposure of their receptive fields to noxious levels of heat. The average magnitude of sensitization was quantitatively similar to that observed in vivo, suggesting that blood-born factors are not essential for development of sensitization. 3. Sensitization in one-half of randomly selected CPNs was blocked or reduced when the perfusate contained a cyclooxygenase inhibitor, indomethacin or dipyrone, or the dual cyclooxygenase/lipoxygenase inhibitor, BW755C, even though initial responsiveness to heat and pressure was unaltered. These observations suggest that arachidonic acid breakdown products, possibly prostaglandins, are intermediaries in the sensitization of some, but not all, C-fiber nociceptors of the skin. In addition, heat-induced sensitization for some C-fiber cutaneous nociceptors is the result of processes that are at least partially independent of those involved in excitation. 4. Substance P (SP) or the putative SP antagonists, [D-Pro2, D-Trp7.9]-SP or [D-Pro2, D-Phe7, D-Trip9]-SP, produced no significant effect on heat-responsiveness or sensitization, although ongoing activity may have marginally increased over control levels after repeated heat stimulations. We conclude that SP in an in vitro preparation is not involved in the enhancement of cutaneous C-fiber nociceptor responsiveness after repeated thermal insults.