It is customary to submit only one portion of a breast cancer to determine if there is amplification or overexpression of the proto-oncogene HER-2/neu. In routine studies of the expression of neu in breast cancer, however, we noted discrepancies in intratumoral positivity. To investigate this phenomenon further, multiple tumor specimens (129 samples) from 41 women with breast cancer were examined. Forty cases were analyzed for neu amplification by slot blot assay and 18 with fluorescent in situ hybridization. Neu overexpression was determined using four different specific antibodies. In more than 50% of cases there were discrepancies in results between the tissue blocks examined. This was evident in both inter- and intra-assay comparisons. It is concluded that intratumoral heterogeneity of neu amplification/overexpression in breast cancer exists to a far greater degree than previously recognized and could be a responsible factor for conflicting published data regarding neu's prognostic significance. Examination of only one tumor sample may not give a true indication of either amplification or overexpression of this oncogene.