The neuropeptide substance P (SP) is found within a subpopulation of nociceptive afferent nerve fibres and has been shown to be upregulated in a variety of sites following peripheral inflammation. The aim of this study was to investigate the expression of SP within human teeth, both in health and disease, and to seek a correlation between reported pain history and SP expression within pulpal nerves. Coronal pulps were removed from 62 permanent mandibular molars with a known pain history. Teeth were categorised as intact, moderately or grossly carious. Using indirect immunofluorescence, sections were double-labelled for the general neuronal marker protein gene product 9.5 (PGP 9.5) and for SP. Image analysis was then used to quantify the percentage area of PGP 9.5-labelled tissue which was also labelled for SP. Throughout the pulp, the expression of SP was found to be significantly increased with the progression of caries. Furthermore, SP expression was significantly greater in grossly carious painful specimens than in grossly carious asymptomatic specimens. These data would suggest that the expression of SP within pulpal nerves undergoes dynamic changes following caries, which may have an important clinical significance in terms of inflammation and pain experience.