The Drosophila GAGA factor binds specifically to simple repeating d(GA.TC)n DNA sequences. These sequences are known to be capable of forming triple-stranded DNA as well as other non-B-DNA conformations. Here, it is shown that GAGA binds to a d[CT(GA.TC)]22 intermolecular triplex with similar specificity and affinity as to a regular double-stranded B-form d(GA.TC)22 sequence. The interaction of GAGA with triplex DNA cannot, however, stimulate transcription in vitro. The affinity of GAGA for triplexes of the purine motif, such as a d[AG(GA.TC)]22 intermolecular triplex, is significantly lower. The DNA binding domain of GAGA is sufficient for efficient binding to triplex DNA. Based on the reported solution structure of the complex of GAGA-DNA binding domain with double-stranded DNA, a model for its interaction with triplex DNA is proposed in which most of the protein-DNA contacts observed in duplex DNA are maintained, especially those occurring through the minor groove. The higher negative charge of the triplex is likely to have also an important contribution to both the specificity and affinity of the interaction.