Ligands for the epidermal growth factor receptor (EGFR) (e.g. EGF and transforming growth factor alpha, TGF alpha) may be included in membrane preparations from human breast tumors, either complexed or not to EGFR. Assessment of EGFR in human placental membranes (HPM) after preincubation with human EGF (hEGF) or human TGF alpha (hTGF alpha) indicated that the presence of these ligands in a membrane preparations did not affect the apparent number of binding sites, but only resulted in an increased apparent dissociation constant (Kd). To obviate the effect of endogenously bound EGFR ligands in a radioligand binding assay, an acid treatment procedure has been used recently. In the present study we show that acid treatment of mammary tumor membranes does not result in increased EGFR levels but does result in an EGFR enriched membrane preparation by elimination of contaminating cytosol proteins. This however also occurred by washing the membranes with assay buffer at neutral pH. From these experiments we conclude that endogenous ligands, if present in membrane fractions from human breast tumors and occupying EGFR, do not interfere in a multipoint radioligand binding assay.