Metallothionein (MT) and the related metal-binding proteins in tissues of lower vertebrates and invertebrates were isolated by gel filtration chromatography (conventional liquid and high performance liquid chromatography). The metal-binding proteins including MT were detected by determining the metals bound to the proteins by atomic absorption spectrophotometry. This analytical procedure was recommendable as the first step to characterize unknown metal-binding proteins in diverse living bodies. As MT in the lower vertebrates, avian (Japanese quail), reptilian (tortoise), amphibian (8 species of frogs and 2 species of tailed amphibians) and fish MTs were isolated and characterized. The number of isoMTs was different among animals belonging not only to different classes but also to different species in the same family. One of the isoMTs in the avian, reptilian and amphibian MTs was shown to contain one histidinyl residue. Cadmium(Cd)-binding proteins were induced in three species of insect (Insecta, Arthropoda) larvae (midge, fleshfly and silkworm) by loading of Cd. Distribution of Cd in insect tissues was different among the three species though the alimentary canal was commonly the most important tissue for the storage of Cd in all species. Further, Cd in the soluble fractions was bound to inducible proteins of different molecular sizes in the three species. Cd-binding protein was also detected in the waterflea (Crustacea, Arthropoda) after loading of Cd. Cd-binding proteins induced in the earthworm (Oligochaeta, Annelida) by loading of Cd were a mixture of proteins with three different molecular sizes. One of the Cd-binding proteins with an apparent molecular size of 7,000 was shown to have characteristics of MT.