A receptor blotting technique was used to detect SH2 domain containing epidermal growth factor receptor (EGFR) substrates that exhibited differential expression either between normal breast epithelial cells and breast cancer cells or between different human breast cancer cell lines. This identified a 25 kD protein, subsequently identified as Grb2, which was markedly overexpressed in three breast cancer cell lines (MCF-7, MDA-MB-361 and -453) relative to both normal breast epithelial cells and the majority of breast cancer cell lines. Northern blot analysis revealed that 7/19 breast cancer cell lines exhibited more than twofold overexpression of Grb2 mRNA, with overexpression correlating with high expression of erbB receptors. In MCF-7, MDA-MB-361 and -453 cells the overexpression of Grb2 mRNA and protein was accompanied by a small amplification of the Grb2 gene locus. Overexpression of Grb2 correlated with increased complex formation between Grb2 and the hSos-1 Ras GDP-GTP exchange protein. This upregulation of the Ras signalling pathway might modulate the growth factor sensitivity of human breast cancer cells and therefore play a role in tumour progression.