To generate a safe vaccinia Tian Tan (VTT)-based vaccine vector, it is necessary to develop a method to attenuate the virus. A modified VTT (MVTT(2-GFP)) was constructed by replacing the viral M1L-K2L genes with a GFP gene. In comparison to the parental VTT, MVTT(2-GFP) lost its replication capacity in rabbit RK13 and human HeLa cell lines. The life cycle of viral replication was blocked at different stages in these two cell lines as determined by electron microscope examination. MVTT(2-GFP) was less virulent than VTT for 100-fold by measuring mouse body weight loss after intranasal viral inoculation and for 340-fold by determining the intracranial LD(50) value in mice. The foreign GFP gene was stable genetically after 10 rounds of passage in Vero cells. Importantly, MVTT(2-GFP) elicited both humoral and cell-mediated immune responses to the GFP gene in mice. With two intramuscular inoculations of 10(5)PFU virus, the anti-GFP antibody reciprocal endpoint titer reached over 700 as determined by an ELISA. The number of IFN-gamma secreting T cells reached over 350SFU per million splenocytes against a CD8+ T cell-specific epitope of GFP. Collectively, the removal of the M1L-K2L genes is a useful method to generate an attenuated vaccinia Tian Tan vaccine vector.