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Substance P (SP) plays an important role during neurogenic inflammation of dental pulp. The purpose of this study was to use a radioimmunoassay for determining the effect of cavity preparation on SP expression in healthy human dental pulp. Ten pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic reasons. Deep cavity preparation (<1 mm remaining dentine thickness) was performed before extraction in five of these bicuspids. All samples were processed and 125I-SP labeled; SP was quantified by competition assay. The results revealed SP expression in all human pulp samples. Mann-Whitney's U test revealed statistically significant higher expression in pulp from teeth where cavity preparation had been performed compared to control values (p < 0.05). These findings suggest that SP is released during common dental procedures (such as cavity preparation) and its expression may have an important clinical significance in terms of experiencing inflammation and pain.