Screening for puparium formation accelerating activity of neuropeptides and/or analogues belonging to 14 different peptide families revealed the strong activity of members and analogues of the pyrokinin/PBAN (pheromone biosynthesis activating neurohormone) family that all share the common C-terminal sequence, FXPRLamide (X = V, T, S, or G). Both pupariation behaviour and cuticular tanning can be accelerated by a C-terminal pentapeptide fragment composed of only the FTPRLamide sequence. Truncation of the C-terminal sequence to the tetrapeptide TPRLa did not diminish either aspect of the activity. Markedly reduced, but still significant, activity was observed after further truncation to the pyrokinin C-terminal tripeptide. The RLa terminal fragment showed no activity. Thus the C-terminal tripeptide appears to be the active core for pupariation acceleration. The core sequence for a maximum response is the C-terminal tetrapeptide TPRLa. This represents a major difference from the activity profile observed in other pyrokinin assays, in which the C-terminal pentapeptide is required. The C-terminal amide group is also of great importance to pupariation acceleration activity, as LPK acid induces a large drop in threshold activity. Periviscerokinin-2 contains a C-terminal tripeptide sequence (PRVamide) that is quite similar to the pyrokinin C-terminal tripeptide PRLamide and, accordingly, elicits a lower level pupariation acceleration activity. The locust pyrokinin Lom-MT-TV preferentially promotes acceleration of the behavioural over the tanning aspects of pupariation and can therefore, in large measure, provide a means of separating the two aspects. Ligation experiments demonstrated that the effect of the LPK analogues on pupariation behaviour is likely mediated through the CNS, while the action on cuticular tanning is of a peripheral nature.