Odontogenic pain often involves inflammation of dental pulp tissue. Dental pulp is highly innervated with a subpopulation of sensory neurons containing neuropeptides. Substance P, released from afferent fibers (e.g. nociceptors) is associated with the development of neurogenic inflammation. In this study, we tested the hypothesis that irreversible pulpitis is associated with increased activity of peptidergic neurons, as measured by increased pulpal levels of immunoreactive substance P (iSP). We determined in vivo pulpal levels of immunoreactive substance P in human teeth with a diagnosis of normal pulp or irreversible pulpitis using CMA/20 microdialysis probes inserted into vital pulps of 24 teeth from 21 patients. Probes were perfused with a modified Locke-Ringer's buffer and immunoreactive substance P levels in the dialysate were measured using a radioimmunoassay. Mean extracellular levels of immunoreactive substance P were significantly higher (>8-fold) in teeth diagnosed with irreversible pulpitis than immunoreactive substance P levels in dental pulp diagnosed as normal (147.7 ± 34.0 pM versus 18.2 ± 6.2 pM). These observations suggest that biochemical measures of inflammatory mediators exhibit significant change during irreversible pulpitis and may contribute to clinical signs and symptoms.