ER-D5 is a recently identified protein related to estrogen receptors (ER). Generally ER measurement requires fresh frozen tissue and for ER-D5 assay ethanol (E) fixation of the specimen is recommended. We evaluated the possibility of immunocytochemical detection of ER-D5 in routine formalin-fixed (F) sections in 51 breast cancers comparing the results with those obtained in the same specimens using E as fixative. The results of ER-D5 assay were expressed by the staining index (SI) taking values greater than or equal to 5 as positive. In all tumors ER was also assayed by a biochemical method (DCCA). The sensitivity of ER-D5 detection in F was only 33.3%, while the specificity was 94.4%. A lower cut-off value of SI for F sections (greater than or equal to 2) increased the sensitivity to 66.6%, leaving the specificity unchanged. A strong correlation was found between the SI of ER-D5 in E and F (p less than 0.001). The SI of ER-D5 in F sections was also well correlated with ER concentrations (p less than 0.001). These results suggest that immunocytochemical determination of ER-D5 in routine sections may be useful in retrospective studies of hormone dependence in breast cancer.