P190-B Rho GTPase activating protein (GAP) is essential for mammary gland development since p190-B deficiency prevents ductal morphogenesis. To investigate the role of p190-B during distinct stages of mammary gland development, tetracycline (tet)-regulatable p190-B overexpressing mice were generated. Short-term induction of p190-B in the developing mammary gland results in abnormal terminal end buds (TEBs) that exhibit aberrant budding off the neck, histological anomalies, and a markedly thickened stroma. Overexpression of p190-B throughout postnatal development results in increased branching, delayed ductal elongation, and disorganization of the ductal tree. Interestingly, overexpression of p190-B during pregnancy results in hyperplastic lesions. Several cellular and molecular alterations detected within the aberrant TEBs may contribute to these phenotypes. Signaling through the insulin-like growth factor (IGF) pathway is altered, and the myoepithelial cell layer is discontinuous at sites of aberrant budding. An increase in collagen and extensive infiltration of macrophages, which have recently been implicated in branching morphogenesis, is observed in the stroma surrounding the p190-B overexpressing TEBs. We propose that the stromal response, disruption of the myoepithelial layer, and alterations in IGF signaling in the p190-B overexpressing mice impact the TEB architecture leading to disorganization and increased branching of the ductal tree. Moreover, we suggest that alterations in tissue architecture and the adjacent stroma as a consequence of p190-B overexpression during pregnancy leads to loss of growth control and the formation of hyperplasia. These data demonstrate that precise control of p190-B RhoGAP activity is critical for normal branching morphogenesis during mammary gland development.