In a multisubtype approach to HIV-1 vaccination, mice were immunized with HIV-1 envelope gp160 genes from subtypes A, B, and C. Subsequently the mice were challenged with syngeneic primary splenocytes infected with a HIV-1/MuLV pseudovirus carrying a subtype B genome. HIV-specific immune responses and protection were strongest in the group of animals immunized with a combination of subtype A, B, and C specific gp160 genes as compared to subtype B only. Immunization with the combination of the cross-reactive subtypes A and C envelope genes induced HIV-specific immune responses but did not result in significant protection to challenge with subtype B infected cells. From this we conclude that immunization with the envelope genes from several HIV-1 subtypes may indeed enhance immune responses. This study shows that by using a mix of subtype envelope genes, an enhanced protective immunity can be obtained experimentally, potentially also in humans.