Abstract An in situ hybridization method, using digoxigenin labelling, for the detection of c-erbB-2 amplification in breast cancer is described. 16 primary breast carcinomas were selected for study. Nuclear hybridization signal was observed in nine cases with amplification detected by both Southern and slot blotting and in only one of seven cases without detected amplification. The hybridization reaction was punctate and multifocal in tumour cell nuclei, suggesting multiple sites of gene copy and transcriptional activity in the nucleus. The hybridization reaction was negative in three cases in which overexpression of oncoprotein (detected by immunocytochemistry and ELISA) occured without amplification. The method described is a reliable means of detecting c-erbB-2 amplification. The high sensitivity and specificity of the reaction and its use in a tissue based system will allow study of a range of possible precursor lesions of breast cancer for evidence of c-erbB-2 amplification.