Objective To construct human histone H2A.X (H2A.X) and nucleophosmin (NPM1) mammalian two-hybrid vectors and to realize their expression in MCF-7 cells. Methods H2A.X and NPM1 coding sequences were amplified by PCR and cloned into pBIND and PACT vectors via TA cloning and validated by restriction enzyme mapping and sequencing. MCF-7 cells were then transfected with H2A.X-pBIND and NPM1-PACT. Overexpression of H2A.X and NPM1 was detected using RT-PCR method . Results PCR amplified the expected H2A.X and NPM1 coding sequence fragments. Restriction mapping of TA, p-BIND and PACT clones produced the predicted DNA bands. Sequencing showed the two vector constructs to completely align with H2A.X and NPM1 sequences. mRNA levels of H2A.X and NPM1 were increased in cells transfected with H2A. X-pBIND and NPM1-PACT, respectively. Conclusions Human H2A. X and NPM1 mammalian two-hybrid vectors were successfully constructed and expressed in MCF-7 cells.