Monocytes were purified from canine peripheral blood by subjecting Ficoll-Hypaque-separated mononuclear cells to either complement-dependent cytolytic treatment with a panlymphocyte antibody (DLy-6) or rosette formation with human red blood cells (RBCs) followed by adherence to fetal-calf-serum-coated plastic dishes, or sedimentation over discontinuous Percoll gradients. These techniques resulted in monocyte enrichment of 83 +/- 3%, 89 +/-6%, and 42 +/- 3%, respectively. Monocytes purified by either the monoclonal antibody method or rosette formation with human RBCs failed to confer concanavalin-A responsiveness to lymphocytes depleted of accessory cells by discontinuous Percoll gradients. The most potent accessory activity was present among cells from low-density fractions of Percoll gradients and populations that did not form rosettes with human RBCs (monocyte-depleted), indicating that cells other than monocytes are involved in accessory function.