Repressor-activator protein 1 (RAP1) has an essential role in the maintenance of yeast telomeres. Yeast telomeric DNA consists of simple repeated G-rich sequences that are bound by RAP1. We have found that RAP1, in addition to its known binding activity for double-stranded DNA, interacts with the G-rich strand containing guanine base (G)-tetrads. We show here using circular dichroism spectroscopy that RAP1 promotes the formation of one particular type of DNA quadruplex, parallel G4-DNA. Furthermore, RAP1 is able to bind to both preformed parallel and antiparallel DNA quadruplexes. These results have implications for the possible use of DNA quadruplexes in telomere-telomere association in vivo.