Objective Root canal treatment (RCT) is often initiated upon pain caused by inflammation of pulpal or periapical tissues. To explore to what extent certain clinical signs and symptoms differ between these two conditions, we compared pain of pulpal and periapical origin with regard to symptoms, clinical signs and diagnostic findings. Methods 62 dentists within the National Dental PBRN (www.NationalDentalPBRN.org) enrolled consecutive patients requiring RCT. Preoperative data were collected from patients (symptoms, pain characteristics) and dentists (examination findings) by questionnaires. Patients with average pain intensity >0 (0–10 numeric rating scale; NRS) for >1 day in the week preceding treatment were eligible. The definition for pain of pulpal origin was tooth pain with bleeding within the pulp chamber in the absence of periapical radiolucency. The definition for pain of periapical origin was tooth pain without bleeding pulp and presence of radiolucency. Pearson's chi-square test and t-test compared groups, and sensitivity and specificity were calculated. Results 370 patients met criteria; 234 (63%) exhibited pulpal pain and 136 (37%) had periapical pain. Patients with pain of pulpal origin more often reported sharp (p=0.004), dull (p=0.039), or shooting pain (p=0.026), provoked pain onset (p=0.002), pain aggravated by cold/hot food or drink (p<0.001) and prolonged response to cold (p<0.001). Patients with pain of periapical origin more often had tenderness to percussion (p<0.001) and biting pressure (p<0.001), as well as swelling (p<0.001). To identify pain of pulpal origin, prolonged response to cold had 71% sensitivity and 92% specificity. To identify pain of periapical origin, tenderness to percussion had 89% sensitivity and 38% specificity and for biting pressure, 81% sensitivity and 42% specificity. Conclusion Clinical characteristics and symptoms clearly differed between conditions. Although no single factor had adequate validity to be used as an indicator of the source of pain, several should be considered for inclusion in future diagnostic algorithms.