To determine if intracellular translocation of the intact insulin molecule was necessary for insulin stimulation of enzyme synthesis, the effect of ng.tive insulin and insulin coupled to Sepharose on tyrosine transaminase levels in cultured rat liver cells was studied. After cells had been exposed to dexamethasone for 18 hr, the addition of crystalline insulin caused a marked increase in tyrosine transaminase levels over the next 2 hr. Insulin was covalently linked to Sepharose beads via the B-29 lysine group by the method of Cuatrecasas and washed extensively. Three separate preparations by three different people in two independent laboratories were made. Free insulin, measured by both immunoassay and bioassay, leaked off of the agarose beads in all three preparations, commencing shortly after the washing was completed and continuing progressively. Although Sepharose—insulin prepartions may still be useful to isolate soluble insulin binding proteins, they are not a suitable model with which to investigate the metabolic effects of bound insulin as long as free insulin can be demonstrated to leak off of the agarose bead. (Endocrinology92: 1442, 1973)