Binding of [3H]Ro5-4864, a ligand selective for peripheral-type benzodiazepine receptors, to human and guinea pig lung membranes was characterized and the binding sites localized in lung sections by light microscopic autoradiography. Inhibition of [3H]Ro5-4864 binding by unlabeled Ro5-4864, PK11195, midazolam, diazepam, clonazepam and flumazenil indicated that [3H]Ro5-4864 bound to specific receptors and binding was reversible. Scatchard analysis indicated a single class of binding site, with a dissociation constant (Kd) of 12.8 +/- 2.1 nM with maximum binding capacity of 2.6 +/- 0.6 pmol/mg of protein for human lung and Kd of 8.4 +/- 1.5 nM and maximum binding capacity of 3.5 +/- 0.3 pmol/mg of protein for guinea pig lung. Autoradiograms showed specific labeling over discrete structures in both human and guinea pig lung. Labeling was particularly dense over submucosal glands in intrapulmonary bronchi of human. Airways were also labeled, with epithelium having a higher grain density than smooth muscle. Labeling over smooth muscle was denser in small compared to large airways. Vascular smooth muscle was not labeled in either human and guinea pig. The alveolar walls were uniformly labeled in both species. The functional significance of pulmonary peripheral-type benzodiazepine receptors remains to be determined.