The gene for the steroid receptor coactivator amplified in breast cancer 1 (AIBI), located on chromosome 20q12, is overexpressed at the mRNA level in up to 60% of primary breast carcinomas; however, only 5% of these tumors show DNA amplification. The transcription factors and signaling pathways relevant to breast cancer, which in the absence of DNA amplification are responsible for and targeted by elevated levels of AIBI mRNA, are unknown. In the present study, in situ hybridization was used to examine AIB1 mRNA expression in 93 breast carcinomas of varying histological grade and immunohistochemical profile. AIB1 mRNA was overexpressed relative to normal breast tissue in 26 of 83 (31%) invasive tumors. This was found to associate with high tumor grade (P = 0.0006), lack of immunohistochemical staining for the steroid receptors estrogen receptor (P = 0.002) and progesterone receptor (P = 0.002), and strong protein staining for p53 (P = 0.01) and HER2/neu (P = 0.002). These findings suggest that AIB1 overexpression may impact on breast cancer by a mechanism not wholly dependent on steroid receptor coexpression and which may involve other oncogenic events, such as p53 protein stabilization and HER2/neu overexpression.