Using a monoclonal antibody specific for the hamster fibronectin receptor (FnR), we have demonstrated that a portion of the CHO cell FnR population is constitutively endocytosed. Three independent techniques were used to study the internalization: 1) after saturation binding of an anti-FnR antibody (PB1) to cells at 4 degrees C, internalization was initiated by warming to 37 degrees C, and then acid/salt elution of membrane-bound ligand was used to quantitate the internalized 125I-PB1; 2) cell vesicular traffic was pharmacologically disrupted with monensin or chloroquine, and the subsequent reduction of the cell surface pool of FnR was monitored; and 3) selective immunoprecipitation was used to separate surface and internalized 125I-labeled FnR. These experiments indicate that about 30% of the cell surface FnR is endocytosed with a t1/2 of 7 min and that this internalization occurs regardless of the ligation state of the receptor. Other observations indicate that the larger fraction of the cell surface FnR pool (70-75%) is apparently shed from the cell upon ligation with antibody at 37 degrees C. This process occurs much more slowly than receptor internalization and leads to an overall reduction in the amount of cell surface FnR. Our results suggest physically or chemically distinct populations of FnR, one of which is unavailable for internalization and recycling.