Gene therapy for HIV (human immunodeficiency virus) involves the introduction of a therapeutic gene into the infected individual for the purposes of reducing viral load and ultimately reconstituting a healthy immune system. Clinical trials for HIV gene therapy have not yet reported therapeutic benefit. In addition to improving the efficiency of gene delivery and the maintenance of gene expression, better therapeutic genes must be designed before this therapy becomes available to patients. A new class of therapeutic genes expressing nucleases may be designed. These nucleases may be classified into three categories based on their mode of action: (i) 'targeted nucleases' for specifically cleaving HIV RNA within the cell, (ii) 'colocalized nucleases' for cleaving HIV genomic DNA or RNA present within the cell or progeny virus, and (iii) 'cytotoxic nucleases' for conferring selective toxicity to HIV-infected cells. The focus of this review is on the design and application of these nucleases for HIV gene therapy.