A recently described protein, found in the haemolymph of Manduca sexta larvae, stimulates ecdysone synthesis by both larval and pupal prothoracic glands in vitro. The mode of action of this haemolymph stimulatory factor has been investigated, particularly as it compares to the action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH). Unlike PTTH, the haemolymph factor does not stimulate ecdysone synthesis via an increase in the level of cAMP in the prothoracic glands. The haemolymph factor requires extracellular calcium for maximal stimulation of the prothoracic glands, but in contrast to PTTH, significant activity is retained in calcium-free medium. Exposure of the prothoracic glands to the haemolymph factor results in enhanced steroidogenesis within 1 min. This rapid stimulation contrasts with the 10-20 min lag period observed following PTTH exposure. However, the prolonged activation elicited by brief exposure to PTTH is not observed following exposure of the glands to the haemolymph stimulatory factor. Rather, the factor appears to be required as a sustained stimulus in order to exert its steroidogenic effects. The data indicate that the mode of action of the haemolymph factor is distinctly different from that reported previously for PTTH, and are consistent with the hypothesized role of the factor as a carrier of a sterol precursor utilized in ecdysone synthesis. © 1986.