A recently isolated haemolymph protein appears to be an important regulator of ecdysone biosynthesis by prothoracic glands in Manduca sexta. Using a dose-response titration protocol, the haemolymph titre of this stimulatory factor was determined during the last larval instar. The titre was high (greater than 2.0 U ml-1) on days 0 and 1, then dropped significantly to 0.55 U ml-1 on day 2, and remained depressed until day 4. The titre of the stimulatory factor then increased to a peak of 1.62 U ml-1 on day 7, and remained elevated (approx. 1.1 U ml-1) until the end of the instar. A set of physical and biochemical criteria was used to confirm that the stimulatory activity present in haemolymph on different days of the instar represented the presence of the factor. The data are consistent with the hypothesis that fluctuations in the titre of the haemolymph stimulatory factor play a critical role in regulating ecdysone biosynthesis during larval-pupal development.