The EMS1 gene encodes an 80185 kDa C-src substrate and localises with the CCNDl gene to chromosome I lq13. This locus is amplified in approximately 13% of human breast cancers. E M S I gene amplification and expression were charactensed in a panel of human breast cancer cell lines to determine at what levels expression is regulated. The degree of tyrosine phosphorylation of EMS1 protein was also determined and compared with the activity of src-family kinases. The EMS1 gene was amplified in 6 of 20 cell lines investigated MDA-MB-134, -157, -I75,453,ZR-75-I and MCF-7. In the MDA-MB-I57 and MCF-7 cell lines, EMS1 was amplified in the absence of CCND I gene amplification. EMS I protein levels were increased relative to normal breast epithelial cells in 6 cell lines (ZR-75-I, MDA-MB-134, -175. 453, MCF-7 and BT-474). Of these, BT-474 is the only cell line that does not exhibit E M S I amplification or increased EMS1 mRNA levels. EMS1 tyrosine phosphorylation was 3-fold higher in BT-474 and T-47D cells, which exhibited relatively high total src activity coupled with expression of both c-fyn and c-yes, than in MDA-MB-453 cells, which expressed only c-yes. Our results therefore demonrtrate gene amplification to be the predominant mechanism underlying EMS1 over-expression in human breast cancer cell lines and identify tyrosine phosphotylation as a further level at which regulation of this protein may be perturbed.