Using complementary DNA microarrays we compared the expression patterns of the osteosarcoma cell line U2OS and three derived subclones obtained by transfection of MDR1, NEO and MDR1/NEO and selected with doxorubicin and neomycin. The expression arrays included 8,976 sequence-verified clones comprising both known genes and expressed sequence tags. We performed Northern blots to confirm the expression changes obtained for certain genes. MDR1 expression was associated with changes in the expression of genes related to different processes, including regulation of certain cell adhesion genes such as zyxin; drug transport proteins such as MRP1; metabolic enzymes such as monoamine oxidase A; apoptotic signaling genes such as I-TRAF; and tumor suppressor genes such as KISS-1. The presence of MDR1 and downregulation of MRP1 were also confirmed by immunohistochemistry. Cells transfected with MDR1 alone or co-transfected displayed more related expression profiles than cells transfected only with the NEO gene, as confirmed by hierarchical clustering. This study provides further characterization of genetic events associated with MDR1 expression, in addition to identifying new targets and potential pathways involved in the molecular mechanism of multidrug resistance.