Fat body of adult male Blaberus discoidalis cockroaches exposed to B. discoidalis hypertrehalosemic hormone (HTH) in vitro showed a decline in tissue glycogen as carbohydrate increased in the medium. In vivo HTH injections increased hemolymph carbohydrate and fat body glycogen phosphorylase activity > 2-fold compared to controls. In vivo trehalose synthesis was unaffected by agents that enhance intracellular levels of cyclic nucleotides including: dibutyrl cAMP, dibutyryl cGMP, forskolin (adenylyl cyclase activator) and isobutyl methylxanthine (IBMX) or theophylline (cAMP phosphodiesterase inhibitors). DbcAMP+IBMX stimulated trehalose biosynthesis of fat body in vitro and had additive effects with a minimally stimulatory HTH concentration. However, adenylyl cyclase activity was unaffected by HTH either with isolated fat body or fat body membrane preparations. We conclude that cAMP is not a second messenger for HTH, but cAMP can stimulate trehalose production independent of HTH through actions on common regulatory events related to trehalose biosynthesis. Dibutyryl cGMP and phorbol esters and diacylglycerol (activators of protein kinase C) also failed to stimulate trehalose biosynthesis in vitro. Extracellular Ca2+ enhanced HTH-dependent phosphorylase activity, glycogenolysis and hypertrehalosemia and maintained basal levels of phosphorylase a at twice those observed in the absence of Ca2+. However, Ca2+ entry by Ca2+ ionophore (A23187) did not mimic HTH effects. Results of these studies demonstrated that extracellular Ca2+ is important for HTH-dependent trehalose biosynthesis but cAMP and cGMP are not involved.