Our laboratory has demonstrated that conjugating the complement protein, C3d, to an antigen is an effective method for eliciting high titer, long-lasting antibodies. The use of C3d as a molecular adjuvant is quite effective in enhancing the efficacy of DNA vaccines expressing the envelope (Env) protein of the human immunodeficiency virus (HIV). However, our previous studies have examined the effectiveness of C3d conjugated vaccines in BALB/c mice. In this study, one outbred strain and three different inbred strains of mice were vaccinated with plasmids expressing a secreted form of Env (sgp120) fused to two or three copies of the murine homologue of C3d (mC3d). Analyses of titers demonstrated that sgp120 conjugated to multiple copies of C3d induced similar high titer anti-Env antibodies in all mouse strains tested. However, the immunoglobulin subclass elicited in inbred mice vaccinated with sgp120-mC3d-DNA was predominately IgG(1), but was a mixture of IgG(1) and IgG(2a) in vaccinated outbred mice. Also, splenocytes from all sgp120-mC3d-DNA vaccinated inbred mouse strains secreted primarily interleukin-4 (IL-4) indicating a T helper (Th) type 2 response. In contrast, mice vaccinated with sgp120-mC3d-DNA had splenocytes that secreted both IL-4 and interferon-gamma (INF-gamma) indicating a mixed T helper response. In addition, the avidity maturation of the anti-Env antisera was enhanced in outbred mice with DNA expressing sgp120-mC3d. Overall, C3d conjugated DNA vaccines elicited enhanced immune responses in outbred mice compared to inbred mice.