Insect prothoracic glands are regulated by neuropeptide prothoracicotropic hormones (PTTH). In Manduca sexta PTTH exists as two size variants, big PTTH (∼25.5 kDa) and small PTTH (∼7 kDa). Previous studies indicate that both size variants employ cAMP as a second messenger and that stimulation of ecdysteroid secretion by big PTTH is Ca2+-dependent. in the present study, experiments were performed to assess the role of Ca2+ in small PTTH-stimulated ecdysteroid secretion by prothoracic glands from fifth instar larvae. Basal ecdysteroid secretion was not affected by Ca2+ channel blockers (verapamil or lanthanum) or by omission of Ca2+ from the incubation medium. Treatment of glands with a Ca2+ ionophore (A23187 or ionomycin) produced a concentration-dependent stimulation of ecdysteroid secretion. Stimulation of ecdysteroid secretion by small PTTH was suppressed (1) by Ca2+ channel blockers and (2) in Ca2+-free medium. A cAMP analog (Sp-cAMPS) stimulated ecdysteroid secretion in the presence of a Ca2+ channel blocker (verapamil) and in Ca2+-free incubation medium, and ionophore-induced ecdysteroid secretion appeared to be suppressed by a cAMP antagonist (Rp-cAMPS). The combined results indicate that basal ecdysteroid secretion is not dependent on external Ca2+, and suggest that small PTTH-stimulated ecdysteroid secretion is mediated by an influx of Ca2+ that precedes cAMP formation. © 1995.