Tissue microarray technology facilitates rapid assessment of expression of molecular markers by enabling the simultaneous analysis of hundreds of tissue specimens. We have applied this technology to establish a microarray composed of cell pellets derived from 40 human lymphoma/leukemia-derived cell lines harboring a variety of molecular abnormalities. The application of cell line microarrays to the assessment of biologic marker evaluation was validated by studying the immunohistochemical expression of PTEN and phosphorylated AKT, two mediators of the phosphatidylinositol (PI)-3-kinase pathway. In addition to the high throughout analysis of protein expression in lymphoma/leukemia cells, this methodology also enables the evaluation of subcellular localization of protein expression. Cytoplasmic PTEN expression was observed in the majority of cell lines (87%), whereas a minor subset demonstrated nuclear expression. Phosphorylated AKT was also expressed predominantly within the cytoplasm in 65% of cell lines, whereas nuclear expression was seen in a minority. An inverse relationship between PTEN and phosphorylated AKT was observed in 63% of cell lines. No cell lines showed absence of PTEN expression, whereas 50% of cell lines showed low PTEN expression. Our data support the integrity of the PI-3-kinase-PTEN-AKT pathway in a majority of cell lines derived from hematologic malignancies and clearly demonstrates the utility of microarray technology in the in situ assessment of expression of molecular markers in tumor-derived cell lines.