Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10−10–10−8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10−9–10−8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10−8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10−10 to 10−8 M to reach its maximum at concentrations of 10−8–10−7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.