Whether B7-1 and B7-2 have distinct functions for eliciting immune responses to antigens that are presented to the immune system by intracellular and extracellular antigen processing pathways is an unresolved question. To investigate this issue we compared the humoral and cellular immune responses elicited by immunizing wild-type, B7-1-/- and B7-2-/- mice with either HIV-1 gp120 plasmid DNA, recombinant gp120 protein or vaccinia virus expressing gp120. The generation of both humoral and cellular immune responses to an antigen produced intracellularly following DNA vaccination had critical requirements for B7-2, but not B7-1. Neither of the molecules was essential for the generation of antibody responses to an extracellular protein antigen administered with adjuvant; B7-1 had little effect on the elicited immune responses. When recombinant vaccinia virus was used to present antigen intracellularly in the context of a viral infection, B7-2 was absolutely required for antibody and T cell proliferative responses, but it exerted a suppressive effect on the elicited CTL activity. These results demonstrate that antigens presented to the immune system by different mechanisms have distinct B7-1 and B7-2 co-stimulatory requirements.