The neuroendocrine light green cells (LGCs) control body growth and metabolism of the freshwater snail, Lymnaea stagnalis. These cells, located in the cerebral ganglia, use the peripheries of the median lip nerves (MLNs) as the neurohemal area. The LGCs express four members of a gene family encoding the precursors of distinct though related molluscan insulin-related peptides (MIPs). The molecular characterization and biosynthesis of MIPs have been investigated. MLN extracts were first size fractionated using gel permeation chromatography, and a major peak that coeluted with bovine insulin was further resolved using reverse phase high performance liquid chromatography. Amino acid composition analysis revealed that the four MIPs were present, as encoded by the MIP genes. Biosynthesis and axonal transport of the MIPs were studied in vitro with LGC systems in a series of pulse-chase incubations in combination with gel permeation chromatography analysis. During the pulse period of 20 min MIP precursors were formed in the LGC cell bodies. During the subsequent chase period these precursors were converted to MIPs and C peptides, which were transported to the LGC axon terminals in the MLNs for storage and release. The MIP precursors seem to be processed similarly to the insulin precursor in pancreatic islets.