We evaluated the compound imiquimod as a possible adjuvant for DNA immunization against human immunodeficiency virus (HIV). We found that gene-gun epidermal delivery of the DNA in combination with imiquimod resulted in the strongest HIV specific immune responses. The effect of imiquimod was further compared to that of recombinant granulocyte macrophage-colony stimulating factor (GM-CSF), a known DNA vaccine adjuvant. Both adjuvants were able to enhance the immune responses induced by the HIV-1 genes alone. The delivery of an adjuvant as a topical cream rather than through injections has a clear clinical benefit. We show for the first time that imiquimod can act as an adjuvant for DNA vaccination.