The presence of ecdysteroids during the larval life cycle and in the abdomens and ovaries of pregnant tsetse flies was established using radioimmunoassay. The hormone titre rises on day 9 of the larval life cycle 2–3 days after the hardening of the polypneustic lobes, mouthparts, phragma and anus. How initiation of tanning of the cuticle of the polypneustic lobes and later the puparium of tsetse fly is regulated is discussed as these events are separated in time by several days. It would seem to be unlikely that the pyrokinin neuropeptide, which is responsible for initiating the events that lead up to pupariation, is also involved 2–3 days earlier in the life cycle. The objective was to ascertain whether 14C-l-p-tyrosine enters the lobe cuticle in vivo and in vitro much faster than into integument cuticle on the 7th day of the pregnancy cycle. The result showed that the uptake of tyrosine into the lobes was 11–12 times faster than into the rest of the cuticle. Some evidence is presented that haemocytes underlying the epidermis are involved in overcoming the barrier to uptake of the phenolic compounds required for sclerotization of the lobes. Pupariation could be retarded by physical means such as ligaturing of the larvae. To try to break this inhibition (retardation) of pupariation in ligatured larvae, 14C-l-p-tyrosine metabolites were injected, with and without ecdysteroids, into the anterior of ligated larvae. The result of this experiment showed that the steroid hormone did not overcome the inhibition but neither does injecting tyrosine, dihydroxyphenylalanine, dopamine or tyramine. Evidence obtained from chromatography and electrophoresis indicated that N-acetylation of dopamine did not occur in the haemolymph of larvae that were artificially retarded. This impact raises the possibility that N-acetyltransferase and acetyl-coenzyme A could be released in pre-pupae after the action of a neuropeptide breaks the retardation of tanning.