Multiple forms of the enzymes exhibiting esterolytic activity have been studied in the tissues of a wide variety of invertebrate and vertebrate species. After investigating the variables in the formation of polyacrylamide gels a rigid set of conditions was identified which enabled the molecular weights of many of these multiple forms to be measured following electrophoresis. Those esterases which had not been previously classified, were tested for substrate and inhibitor specificity, their physicochemical properties established and their relationships to existent tabulations established. Ontogenetic and genetic studies were carried out on the esterases of the trout and turtle tissues, and from these evidence was derived to support the proposal that polymorphism exists in the tissues of the trout. Immunoelectrophoresis and complement fixation inhibition assays were carried out in a number of vertebrate tissues using antisera produced against purified esterases. The former technique established relationships existing between groups of multiple forms or similar specificity in a wide variety of tissue types. Complement fixation inhibition to a significant degree was established between a carboxylesterase and a cholinesterase. The significance of these findings has been discussed in connection with the compositional and phylogenetic interrelationships of the animal esterases.