Previous studies in our laboratory have characterized non-conventional opioid binding sites in membrane preparations from both rat and human lung. The studies described in this paper utilized autoradiography to investigate the regional distribution of these [3H]morphine binding sites within rat lungs. Specific binding of [3H]morphine was saturable and Rosenthal analysis of tissue section wipes revealed the presence of both high-affinity and low-affinity opioid binding sites. The mean +/- S.E.M. binding affinity and the mean +/- S.E.M. density values for the low-affinity binding site (Kd = 217 +/- 160 nM, Bmax = 12 +/- 8 pmol/mg protein) were similar to the values obtained in our previous whole-rat lung membrane binding assays (Kd = 187 +/- 36 nM, Bmax = 13.5 +/- 2 pmol/mg protein) (Cabot, P.J., P.R. Dodd, T. Cramond and M.T. Smith, 1994, Eur. J. Pharmacol. 268, 247). Quantitative autoradiography showed that the highest density of opioid binding sites appeared to be present within the alveolar wall (13.2 +/- 0.8 pmol/mg protein). A significantly lower (P < 0.05) density of binding was also observed in the smooth muscle of the trachea and main bronchi (5.5 +/- 2.1 pmol/mg protein). However, no morphine binding sites were evident in the smooth muscle surrounding the smaller airways and pulmonary vasculature within the lobes of the rat lung. It remains to be investigated whether the opioid binding sites located within the trachea and main bronchi of the rat airways are the prejunctional opioid receptors on C-afferent nerve fibres which modulate the release of potent inflammatory neuropeptides.