The steroid hormone 20-hydroxyecdysone (20-HE) regulates several processes during insect metamorphosis. We studied the effects of 20-HE on the development of voltage-sensitive ionic currents of thoracic leg motoneurons of Manduca sexta. The larval leg motoneurons persist throughout metamorphosis but undergo substantial morphological reorganization, which is under the control of 20-HE and accompanied by changes in Ca2+ and K+ current densities. To determine whether 20-HE controls the changes in Ca2+ and K+ current levels during postembryonic development, identified thoracic leg motoneurons isolated from late larval and early pupal stages were taken into primary cell culture. Whole-cell Ca2+ and K+ currents were measured after 1-4 days of steroid hormone incubation. In the presence of 20-HE, peak Ca2+ currents of pupal leg motoneurons increased from day 1 to day 4 in vitro. Thus, at culture day 4 the pupal Ca2+ current levels were larger in 20-HE-treated than in untreated cells. By contrast, 20-HE did not affect the Ca2+ current amplitudes of larval leg motoneurons. Whole-cell K+ currents, measured at 4 days in pupal motoneurons, consisted of a fast-activating transient current and a sustained, slowly inactivating current. 20-HE did not affect the amplitude of the transient or sustained currents after 4 days in vitro. Thus, a direct steroid hormone effect may control the proper maturation of voltage-sensitive Ca2+ currents in leg motoneurons.