Pre-ecdysis- and ecdysis-triggering hormones (PETH and ETH) from endocrine Inka cells initiate ecdysis in moths and Drosophila through direct actions on the central nervous system (CNS). Using immunohistochemistry, we found Inka cells in representatives of all major insect orders. In most insects, Inka cells are numerous, small and scattered throughout the tracheal system. Only some higher holometabolous insects exhibit 8-9 pairs of large Inka cells attached to tracheae in each prothoracic and abdominal segment. The number and morphology of Inka cells can be very variable even in the same individuals or related insects, but all produce peptide hormones that are completely released at each ecdysis. Injection of tracheal extracts prepared from representatives of several insect orders induces pre-ecdysis and ecdysis behaviours in pharate larvae of Bombyx, indicating functional similarity of these peptides. We isolated several PETH-immunoreactive peptides from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and identified the gene encoding two putative ETHs in the mosquito Anopheles gambiae. Inka cells also are stained with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, our enzyme immunoassays show that these antisera cross-react with PETH and ETH. Our results suggest that Inka cells of different insects produce only peptide hormones closely related to PETH and ETH, which are essential endocrine factors required for activation of the ecdysis behavioural sequence.