Intramuscular injection of plasmid DNA is an efficient method to introduce a foreign gene into a live animal. We investigated several factors affecting the gene transfer efficiency and the following immune response by intramuscular injection of plasmid DNA. When the strength of several highly efficient viral promoters was compared in muscle by using the chloramphenicol acetyltransferase (CAT) gene as an indicator, cytomegalovirus (CMV) immediate early promoter was found to be stronger than any other viral promoters including Rous sarcoma virus (RSV), murine leukemia virus (SL3-3) and simian virus 40 (SV40) early promoters. Inclusion of adenovirus tripartite leader (TPL) sequences and a synthetic intron in the 5' untranslated region of mRNA moderately stimulated the CAT expression. On the other hand, the expression of encephalomyocarditis virus (EMCV) VP1 gene was greatly enhanced by the TPL sequences and an intron. The level of humoral immune response by intramuscular injection of various VP1 expression plasmids was compared. The seroconversion rate was highly dependent on the strength of the expression vector. However, the ratio of IgG1 and IgG2a immune response was not significantly variable depending on the strength of the expression vector. Also, the efficiency of the sindbis virus-based DNA vector was examined for the gene expression and immune response. Although a high level of CAT expression was obtained in muscle by using this system, VP1 was not produced as much as the conventional expression vectors. Furthermore, little humoral immune response was elicited by intramuscular injection of VP1-expressing sindbis vector, suggesting that this system was not superior to the conventional vector for DNA immunization.