Cerebrospinal fluid (CSF) obtained by acute percutaneous puncture of the cisternal membrane of the halothane anesthetized rat has low but measurable concentrations of β-endorphin-like immunoreactivity (β-EPir: 32.8 ± 3.0 pmol/l). Chromatographic separation of β-EPir showed that authentic β-endorphin 1–31 was the main component of β-EPir in cisternal CSF. Subcutaneous injection of 5% formalin in the hind paws did not increase β-EPir in cisternal CSF. Rats with tactile paw hyperalgesia evoked by unilateral ligation of the L 5/6 nerve roots 2 weeks earlier had β-EPir concentrations that did not differ from sham operated or unoperated control animals. In contrast, capsaicin injected in the hindpaws increased the mean β-EPir concentration compared to saline injections (P = 0.006) 45 min after emerging from anesthesia following injection. These results show that acute activation of C fibers (by capsaicin) will evoke the release of β-endorphin into the CSF, suggesting activation of the β-endorphin terminal systems in the brain/midbrain. The failure of formalin injections to release β-EPir to CSF may be due to specificity of the afferent stimulus evoking β-EPir release, a lower stimulus intensity, and/or the duration of the stimulus generated by formalin. The normal concentrations of β-EPir found in the hyperalgesic state following nerve injury suggest that the supraspinal β-endorphin system does not display tonic changes under such conditions.