Abstract Previous studies have shown that the tripeptide Gly-Pro-Ala, a substrate for dipeptidyl peptidase IV (DPP IV, EC 126.96.36.199), interferes with initial spreading of hepatocytes on a matrix consisting of fibronectin and denatured collagen. In the present investigation we report that the tripeptide as well as the anti-DPP IV antibody inhibits the initial spreading of hepatocytes also on native collagen. This effect appears to be due to the interaction of DPP IV from hepatocyte plasma membrane with native collagen. It is shown in vitro by immunohistochemistry, catalytic histochemistry, and by affinity chromatography of solubilized plasma membrane on collagen-Sepharose that DPP IV has a binding affinity to collagen. This binding does not affect the activity of DPP IV.