The PE35 (Rv3872) gene of Mycobacterium tuberculosis is present in the region of difference (RD) one that is deleted in all vaccine strains of Mycobacterium bovis bacillus Calmette Guerin. The aim of this study was to clone PE35 DNA into a DNA vaccine plasmid with CMV promoter and interleukin-2 secretory signal and evaluate the recombinant plasmid for induction of antigen-specific cellular responses in mice. DNA corresponding to PE35 was PCR amplified from the genomic DNA of M. tuberculosis H37Rv, cloned into pGEMT-Easy vector and sub-cloned into the DNA vaccine vector pUMVC6. BALB/c mice were immunized with recombinant pUMVC6/PE35 and spleen cells were tested for T-helper (Th)1-type (antigen-induced proliferation and secretion of IFN-γ) and Th2-type (IL-5), and anti-inflammatory (IL-10) cytokine responses to pure recombinant PE35 protein and its synthetic peptides. Mice immunized with the recombinant plasmid DNA (pUMVC6/PE35) showed positive Th1-type cellular responses to pure PE35, but not to an irrelevant antigen, i.e. PPE68 (Rv3873). However, the vaccine construct did not induce antigen-specific Th2-type (IL-5) or anti-inflammatory (IL-10) reactivity to PE35. Testing with synthetic peptides showed that Th1-type cells recognizing various epitopes of PE35 were induced in mice immunized with pUMVC6/PE35 DNA. These results suggest that pUMVC6/PE35 may be useful as a safer vaccine candidate against TB.