The detection of epidermal growth factor receptors (EGFR) has been proposed as a prognostic factor in different kinds of neoplastic diseases. In this study, we have compared different conditions of EGFR assay, in human placental membranes, in order to establish the best conditions for the routine use of EGFR assay in clinical laboratories. Three kinds of membrane treatment (non-treated, preincubated at 37 degrees C for 30 min, or acid treated at pH 3.0 for 3 min at 0 degrees C), two different separation conditions (hydroxylapatite and centrifugation), two different incubation times (30 min at 37 degrees C and overnight at 18 degrees C) and the effect of proteolytic enzyme inhibitors have been investigated. Preincubated or acid treated membranes showed a two- and three-fold increase of the number of receptors, respectively, as compared with non-treated membranes. In the case of acid pretreated membranes a second, low affinity, site became apparent. Both separation methods gave similar results. The addition of aprotinin had an effect only during long incubation conditions. Freezing of membranes in liquid nitrogen, followed by storage at -80 degrees C for 48 h, resulted in three different patterns. No change was observed in non-treated membranes. Preincubated samples showed a significant decrease both in the number and the affinity of detected EGFR. Acid treated membranes showed a decrease of the affinity of high affinity EGFR with no modification of their number. It is proposed that acid pretreatment, freshly prepared membranes, addition of aprotinin and the use of hydroxylapatite, for the separation of bound and free radiolabelled EGF, should be used in order to standardize the EGFR assay in clinical laboratories. Using these conditions, we were able to detect a significantly higher number of EGFR in 6/29 cases of human breast cancer specimens.