At specific intervals, increased concentrations of two steroid hormones, i.e., ecdysone (E) and 20-hydroxyecdysone (20E), elicit developmental changes in arthropods. Conversion of E to the active molting hormone, 20E, in the tobacco hornworm Manduca sexta is catalyzed by the cytochrome P450-containing ecdysone 20-monooxygenase system (E20M). During embryogenesis, M. sexta E20M activity increased for the first 72 hours at which time it peaked and subsequently significantly declined. The increased activity coincided with the increase of free ecdysteroids and the progression of two embryonic molts. In midgut tissue of fifth instar M. sexta, decreases in second messenger 3’,5’cyclic guanosine monophosphate (cGMP) concentration inhibited day five E20M activity, but increases in cGMP concentration restored E20M activity. Midgut cGMP content peaked on day five of the instar in concert with the highest level of E20M activity observed. Molecular studies with midgut tissue demonstrated that the E agonist RH-5849 elicited increases in E20M (the shade gene) expression. In the presence of two guanylate cyclase inhibitors, E20M expression significantly increased. Inhibition remediation by pharmacological means resulted in significantly decreased shade expression. While it is unclear as to where cGMP exerts its effects on E20M activity, the data indicated that the second messenger affected the level of transcription, translation, or enzyme activity either individually or in some combination. Interestingly, E20M activity also was found to be affected by six synthesized anthraquinones suggesting that these compounds can serve to disrupt M. sexta development. Lastly, E20M-like activity was observed in female Ascaris suum (Nematoda) both in muscle and reproductive tissue. Although E20M localization in muscle was unclear, in reproductive tissue E20M activity resided mainly with microsomes. This work provides a number of important insights into the regulation of M. sexta E20M during development, a role of cGMP in these events, and the possible occurrence of E20M in the parasitic nematode Ascaris suum.