Serum albumin, serum γ globulin, crystalline glucagon and crystalline insulin were observed to bind to diaphragm in vitro and to be eluted slowly on continuous washing with buffered solutions. A lack of reproducibility of binding of insulin at low concentrations is attributable in part to variable adsorption to glassware. A definite saturation of binding sites on diaphragm was not observed at the highest concentrations employed. Competitive inhibition was observed in the presence of heterologous proteins. Binding capacity was not altered by treatment of diaphragms with formaldehyde. Binding of insulin by isolated rat diaphragm in vitro is not demonstrably of biologic significance but is attributable to nonspecific surface adsorption of proteins.