Objective.‐Postlumbar puncture headache may represent a model which could be used to test the hypothesis that headache pain is caused by the release of substance P in patients who are predisposed to headache due to hypersensitivity to substance P.Methods.‐We measured substance P in CSF and plasma in 37 patients undergoing diagnostic lumbar puncture. In 9 patients, plasma samples were obtained before lumbar puncture, in 28 patients plasma was obtained after lumbar puncture. Patients were followed up by telephone to determine if they developed postlumbar puncture headache. Patients were also asked about a history of chronic or recurrent headaches. Substance P was determined by radioimmunoassay.Results.‐The mean plasma substance P levels obtained before lumbar puncture was 1.0 ± 0.1 pg/mL and 1.3 ± 1.2 after lumbar puncture (P<0.0005). The mean plasma substance P levels in subjects who developed postlumbar puncture headache was 0.6 ± 0.6 pg/mL compared with 1.4 ± 1.5 in subjects who remained headache‐free (P<0.05). The mean CSF substance P levels in subjects who developed postlumbar puncture headache was 0.7 ± 0.5 pg/mL compared with 1.2 ± 0.8 in subjects who remained headache‐free (P<0.05). There were no significant differences in substance P levels between chronic headache sufferers and nonheadache subjects.Conclusions.‐Postlumbar puncture headache may be mediated by the release of substance P triggered by lumbar puncture, in patients predisposed to headache by a hypersensitivity to substance P. Hypersensitivity to substance P may also represent a mechanism for headache pain in other h