To determine the influence of DNA sequence on immunostimulatory properties of vaccine vectors, we tested the induction of in vitro and in vivo immune responses by plasmids modified to contain extended runs of dG sequences. Studies with oligonucleotides indicate that dG sequences can directly stimulate B cells as well as enhance the activity of immunostimulatory CpG motifs because of interaction with the macrophage scavenger receptor (MSR); this receptor can bind a variety of polyanions including dG sequences. To modify vectors, we introduced stretches of 20-60 dG residues into the pCMV-beta and pSG5rab.gp vectors and measured the ability of these plasmids to induce IL-12 and IFN-gamma production by murine splenocytes. The induction of in vivo antibody responses to rabies glycoprotein was also assessed with the pSG5rab.gp vectors. In in vitro cultures, cytokine production induced by plasmids with and without dG sequences was similar. Furthermore, the addition of dG sequences to pSG5rab.gp vectors failed to enhance the anti-rabies glycoprotein response to immunization. To assess further mechanisms by which plasmids stimulate macrophages, we measured the effects of MSR ligands on in vitro cytokine induction. In in vitro cultures, poly(G), dG30, and fucoidan inhibited IL-12 induction by plasmids. IL-12 induction was also inhibited by mammalian DNA but was unaffected by polyanions that are not MSR ligands. Together, these results suggest that the addition of 20 to 60-base dG sequences to plasmids does not significantly affect their properties as immunostimulators or vaccines. Furthermore, these results suggest that MSR ligands can block cytokine induction by plasmid DNA whether or not the plasmid contains extended runs of dG.