Colonic absorption of recombinant human insulin-like growth factor I (rhIGF-I) was measured in vitro using both rat and minipig colon. The permeability coefficients were 8.03 ± 1.03 and 4.75 ± 0.43 × 10−8 cm sec−1 in the rat and minipig, respectively. The steady-state flux in rat colon was linearly related to the donor concentration over the range 1 to 10 mg/mL. rhIGF-I was metabolically stable in contact with both mucosal and serosal surfaces of washed colon for 5 hr. The amount of IGF-I permeating through the tissue was quantitated by radioimmunoassay and the identity and integrity of the permeating species were confirmed by reverse-phase HPLC, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and Western blotting. In all cases the permeant was identical to authentic rhIGF-I. The integrity of the colonic tissue in vitro was demonstrated by the maintenance of electrophysiological parameters, a secretory response to serosal theophylline, and the ability of sodium azide, a metabolic inhibitor, to abolish the barrier properties and cause a large increase in flux.