SYNOPSIS. An in vitro assay for the insect prothoracicolropic hormone (PTTH) has been developed which measures the rate of ecdysone synthesized by Manduca sexta prothoracic glands (PG) stimulated in vitro by PTTH. This assay has been used to quantify PTTH in single neurosecretory cells (NSC) resulting in the identification of one NSC in each hemisphere of the brain as the prothoracicotrope, source of PTTH. The axonal and dendritic distribution of the prothoracicotrope has been determined by cobalt filling with silver intensification. From a comparison of the titers of PTTH in brains, corpora cardiaca and corpora allata during larval-pupal development, the corpus allatum has been identifiedas the neurohemal organ for PTTH. Electron microscopic analyses suggest that the acellular sheath surrounding the corpus allatum contains the axon terminals of the prothoracicotropes. There is at least one form of PTTH, ˜22,000 mol wt (big PTTH), and possibly a smaller form of about 7,000 mol wt (small PTTH). Bioassay and PTTH hemolymph titer data during the head critical period (HCP) for larval-larval development reveal that big PTTH is released as a single peak lasting ˜6 hr. By contrast, during the first HCP of the last larval instar PTTH is released over a period of ˜18 hr in three bursts, but its molecular weight has not been established with certainty. The kinetics of PG activation by these two forms suggest that big PTTH may function to activate the PG dramatically and thereby elicit molting, while small PTTH may activate the PG minimally at the time of cellular reprogramming.