Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.