In this study, we analysed gene amplification, RNA expression and protein expression of the c-myc gene on archival tissue specimens of high-grade human breast cancer, using fluorescent in situ hybridisation (FISH), nonradioactive in situ hybridisation and immunohistochemistry. The specific question that we addressed was whether expression of c-Myc mRNA and protein were correlated with its gene copy amplification, as determined by FISH. Although c-Myc is one of the most commonly amplified oncogenes in human breast cancer, few studies have utilised in situ approaches to directly analyse the gene copy amplification, RNA transcription and protein expression on human breast tumour tissue sections. We now report that by using the sensitive FISH technique, a high proportion (70%) of high-grade breast carcinoma were amplified for the c-myc gene, irrespective of status of the oestrogen receptor. However, the level of amplification was low, ranging between one and four copies of gene gains, and the majority (84%) of the cases with this gene amplification gained only one to two copies. Approximately 92% of the cases were positive for c-myc RNA transcription, and essentially all demonstrated c-myc protein expression. In fact, a wide range of expression levels were detected. Statistically significant correlations were identified among the gene amplification indices, the RNA expression scores and protein expression scores. c-myc gene amplification, as detected by FISH, was significantly associated with expression of its mRNA, as measured by the intensity of in situ hybridisation in invasive cells (P=0.0067), and by the percentage of invasive cells positive for mRNA expression (P=0.0006). c-myc gene amplification was also correlated with the percentage of tumour cells which expressed high levels of its protein, as detected by immunohistochemistry in invasive cells (P=0.0016). Thus, although multiple mechanisms are known to regulate normal and aberrent expression of c-myc, in this study, where in situ methodologies were used to evaluate high-grade human breast cancers, gene amplification of c-myc appears to play a key role in regulating expression of its mRNA and protein.