The fruitfly, Drosophila melanogaster, and the earthworm, Annelida oligocheta, were extracted with acid-ethanol by a classic method for recovering insulin from the pancreas. When each extract was filtered on a Sephadex G-50 column, a distinct peak of insulin immunoreactivity (equivalent to 0.1 to 2 ng of insulin/g wet weight) was recovered in the region typical of insulin. The material in this peak had reactivity in the insulin bioassay, measuring stimulation of glucose oxidation or lipogenesis by isolated rat adipocytes. The bioactivity was partially or largely neutralized by anti-insulin antibodies. In concordance with previous work showing the presence of material very similar to insulin in the blowfly and molluscs, we have confirmed the presence of insulin in insects and extended the observation to earthworms. These findings suggest that insulin is more widespread in invertebrates than was previously thought. In a companion study (Proc. Nati. Acad. Sci. USA 77:6184-88, 1980), we have demonstrated material similar to insulin in unicellular organisms.