The aim of this study was to investigate the presence of substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) in the gingival crevicular fluid of teeth diagnosed with pain of pulpal origin compared with clinically healthy teeth, and to detect any changes in the levels of these neuropeptides in gingival crevicular fluid after removal of the pulp from the painful teeth. Gingival crevicular fluid was collected at baseline from one interproximal site at a painful and a non-painful contralateral tooth from 54 adult patients. Sampling was repeated after 1 wk in a subset of 21 subjects. Samples were analysed for SP, NKA, and CGRP using radioimmunoassay. The mean levels of SP and NKA were significantly higher in gingival crevicular fluid from painful teeth compared with non-painful teeth. The level of SP in the GCF of painful teeth fell significantly 1 wk after pulpectomy. In contralateral teeth, there were no significant differences in the levels of SP and NKA after 1 wk. It is concluded that SP and NKA are present in significantly greater amounts in the GCF of painful teeth compared with healthy teeth.