Insulin binding was demonstrated in cultured HT 29 cells originating from a human colon carcinoma. At 37 degrees and in complete medium, the binding of [125I]insulin (1-4x10-10M) reaches a maximum in 40 min and the cell associated radioactivity remains constant for at least 4 h. No degradation of the hormone is observed under these conditions. The binding is proportional to the number of cells and its pH optimum is 7.8. In the presence of excess insulin 50% of the [125I]insulin is dissociated from the complex after 10 min. At equilibrium, insulin binding is specific: proinsulin is 25 times less potent than native insulin in competing with [125I]insulin and related polypeptide hormones are inactive. Scatchard analysis indicates two classes of binding sites (1400 sites/cell of "high affinity" e.g. 4.7 x 108 M-1, and 20 000 sites of "low affinity" e.g. 4 x 107 M-1). The binding of insulin to this non-target cell shows the same kinetic characteristics and specificity as found for insulin in its target cells, except that HT 29 cells do not degrade the hormone. The problem of the correlation between insulin binding and a biological effect in these cells remains to be elucidated.