Immunization with plasmids expressing specific genes (DNA or nucleic acid vaccination (NAV)) elicits robust humoral and cell-mediated immune responses. The mechanisms involved in T cell activation by NAV are incompletely characterized. We have examined the costimulatory requirements of NAV. CD28-deficient mice did not mount Ab or CTL responses following i.m. immunization with eukaryotic expression plasmids encoding the bacterial gene beta-galactosidase (beta gal). Because these mice retained their ability to up-regulate the CTLA4 receptor (a negative regulator of T cell activation), we examined CTLA4's role in the response of wild-type BALB/c mice to NAV. Intact anti-CTLA4 mAb but not Fab fragments suppressed the primary humoral response to pCIA/beta gal without affecting recall responses, indicating CTLA4 activation inhibited Ab production but not T cell priming. Blockade of the ligands for CD28 and CTLA4, CD80 (B7-1) and CD86 (B7-2), revealed distinct and nonoverlapping function. Blockade of CD80 at initial immunization completely abrogated primary and secondary Ab responses, whereas blockade of CD86 suppressed primary but not secondary responses. Simultaneous blockade of CD80 + CD86 was less effective at suppressing Ab responses than either alone. Enhancement of costimulation via coinjection of B7-expressing plasmids augmented CTL responses but not Ab responses, and without evidence of Th1 to Th2 skewing. These findings suggest complex and distinct roles for CD28, CTLA4, CD80, and CD86 in T cell costimulation following nucleic acid vaccination.