Abstract The role of the brain as a neuroendocrine regulator of metamorphosis in the gypsy moth, Lymantria dispar L. was originally demonstrated by Kopec more than 70 years ago. We have reexamined this role by determining haemolymph ecdysteroid titres, activity and responsiveness of the prothoracic glands, and growth of female last (i.e. fifth)-instar larvae in relation to secretion of the brain prothoracicotropic hormone (PTTH), the factor known to trigger moulting and metamorphosis in insects. The head critical period for secretion of PTTH in Lymantria reared at 25°C in a 16 h light-8 h dark cycle was shown by neck ligation to occur on day 7–8 of the fifth instar. The critical period occurred shortly after a small peak of haemolymph ecdysteroid that reached 600 pg 20-hydroxyecdysone equivalents/μl, as determined by radioimmunoassay. Feeding was completed by day 7, thus neck ligation did not affect nutrient intake. Animals starved from day 5 onward completed pupation at the same time as non-starved controls and showed normal increases in haemolymph ecdysteroid titres. Animals neck-ligated on day 7 and injected 5 days later with extracts of brain, retrocerebral complex, or brain tissues containing lateral or median neurosecretory cells were stimulated to pupate within 5 days, and brain-extract-injected animals showed an increase in haemolymph ecdysteroid titre of over 4000 pg/μl. Animals neck-ligated earlier had lower haemolymph ecdysteroid titres than day 7, neck-ligated larvae and their prothoracic glands showed lower activity in vitro . Using the in vivo assay, PTTH activity was detected in pre-hatch eggs and in brains taken from day-5 last-instar larvae, day-1 pupae and day-1 adults. The assay appears specific to tissues from the brain and retrocerebral complex, since no activity was found with extracts of the subesophageal ganglion, fat body or muscle.