The purpose of this investigation was to quantify the concentration of endotoxin in necrotic root canals and investigate the possible relationship between the concentration of endotoxin and endodontic signs and symptoms. Samples were collected from root canals of 50 patients requiring endodontic treatment due to necrosis of the pulpal tissue. Anaerobic techniques were used to determine the number of c.f.u. in each sample. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the concentration of endotoxin in each sample. The presence of c.f.u. was detected by culture in all samples (range 102–5 × 106). In samples from cases of patients with spontaneous pain, the mean c.f.u. was 1.43 × 106 while in asymptomatic cases it was 9.1 × 104. Endotoxin was present in all the samples studied [range 2390.0−22100.0 endotoxin units (EU) ml−1]. The mean concentration of endotoxin in samples from patients with spontaneous pain was 18540.0 EU ml−1 while in asymptomatic cases it was 12030.0 EU ml−1. Asymptomatic cases generally had lower levels of endotoxin (i.e. a negative association). A positive association was found between endotoxin and symptomatic cases (e.g. spontaneous pain, tenderness to percussion, pain on palpation, swelling and purulent exudates). This study showed that endotoxin is present in high concentrations in root canals of symptomatic teeth. There was a positive correlation between the concentration of endotoxin in the root canal and the presence of endodontic signs and symptoms.