The recombinant eukaryotic expression vector pCMV4-rmCGbeta, inserted full-length cDNA of the beta-subunit of rhesus monkey chorionic gonadotropin (rmCGbeta), as DNA immuno-contraceptive against CGbeta glycoprotein, has previously demonstrated the biological expression of rmCGbeta in vitro and in vivo. The plasmid DNA of pCMV4-rmCGbeta was inoculated into BALB/c mice at different doses and routes as DNA immuno-contraceptive to understand its antifertility effect. The results of immune responses indicated that the intradermal inoculation is the optimal pCMV4-rmCGbeta DNA delivery method for BALB/c mice, and the dose of 10 microg should be enough to elicit immune response. With different doses from 10-50 microg, marked reductions in the fertility of the female mice after two intramuscular inoculations of pCMV4-rmCGbeta DNA were seen, while the similar level of humoral immune responses were induced. With the dose of 20 microg of pCMV4-rmCGbeta DNA, the mice showed reduction in fertility from intraperitoneal, and intradermal to intramuscular inoculating method. The antifertility effect of antiserum from immunized mice confirmed that the antibodies elicited by pCMV4-rmCGbeta DNA could prevent pregnancy in female mice. At the same time, the full-length cDNA of beta-subunit of mouse chorionic gonadotropin (muCGbeta) was cloned from placenta and sequenced for the first time (GenBank Accession No. AF333067). Sequence analysis showed that muCGbeta shares 99.6% homology with rmCGbeta and 90.6% with hCGbeta respectively. The results indicated that the infertility of BALB/c mice induced by pCMV4-rmCGbeta contraceptive should be further studied as a CGbeta DNA contraceptive. (Mol Cell Biochem 231: 89-96, 2002)