The biological activity of Sepharose-insulin in different assays in vitro, e.g., stimulation of glucose oxidation, lipogenesis, and antilipolysis and activation of pyruvate dehydrogenase (EC 184.108.40.206) activity, has been investigated. According to amino acid analysis, between 270 and 330 mug (6.9-8.2 U) of insulin were coupled per ml of packed beads. Related to the total insulin content, 0.2-0.7% of the insulin was biologically active. Comparable biological activity was observed with isolated fat cells and fat pad pieces. After incubation with tissue or cells, Sepharose-insulin particles were separated by centrifugation from the medium. The clear supernatant was assayed for biologically and immunologically reactive insulin and contained soluble insulin activity. A quantitative evaluation of the soluble biological and immunological insulin activity in the supernatant accounted for the total insulin activity of Sepharose-insulin.