Tumor cells that are grown in three-dimensional (3D) cell culture exhibit relative resistance to cytotoxic drugs compared with their response in conventional two-dimensional (2D) culture. We studied the effects of targeted agents and doxorubicin on 2D and 3D cultures of human breast cell lines that represent the progression from normal epithelia (modeled by MCF10A cells) through hyperplastic variants to a dysplastic/carcinoma phenotype (MCF10.DCIS cells), variants transformed by expression of activated Ras, and also a basal-subtype breast carcinoma cell line (MDA-MB-231). The results showed the expected relative resistance to the cytotoxic agent doxorubicin in 3D cultures, with greater resistance in normal and hyperplastic cells than in carcinoma models. However, the response to the targeted inhibitors was more complex. Inhibition of mitogen-activated protein kinase kinase (MEK) by either 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) or 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (CI-1040, PD184352) produced a similar inhibition of the growth of all the MCF10 cell lines in 2D. In 3D culture, the normal and hyperplastic models exhibited some resistance, whereas the carcinoma models became far more sensitive to MEK inhibition. Increased sensitivity to MEK inhibition was also seen in MDA-MB-231 cells grown in 3D compared with 2D. In contrast, inhibition of phosphatidylinositol 3'-kinase activity by wortmannin had no significant effect on the growth of any of the cells in either 2D or 3D. Our conclusion is that 3D culture models may not only model the relative resistance of tumor cells to cytotoxic therapy but also that the 3D approach may better identify the driving oncogenic pathways and critical targeted inhibitors that may be effective treatment approaches.