The full-length cDNA encoding the subunits p40 and p35 of human interleukin-12(hlL-12) were cloned separately by RT-PCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus whioa initiates cap-independent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into E1 region of Ad5 genome in cosmid vector pAxlcw of El-substitution type. By homologous recombination with EcoT22I-digested Ad5 DNA-TPC in 293 cells, the replication-deficient recombinant adenoviruses of hIL-12 were generated efficiently. After infected with hIL-12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL-12 at comparable levels (30~60ng/10⁶cells/24hr), which could stimulate the proliferation and IFN-γ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL-12 could effectively mediate the expression of bioactive hlL-12 and might be used in cancer gene therapy.