Abstract The binding of [3H]tyrosyl-PBAN28-33NH2 to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10mM HCO3 ions. Equilibrium of ligand binding was obtained after 20min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a Kd of 5.731.05106M and a Bmax of 1.85 0.22nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH2 and PBAN28-332 with a Ki of 4.31.1106M and 4.92.6106M, respectively.