Background: Chemically induced dimerization (CID) can be used to manipulate cellular regulatory pathways from signal transduction to transcription, and to create model systems for study of the specific interactions between proteins and small-molecule chemical ligands. However, few CID systems are currently available. The properties of, and interactions between, Escherichia coli dihydrofolate reductase (DHFR) and the ligand methotrexate (MTX) meet many of the desired criteria for the development of a new CID system. Results: BisMTX, a homobifunctional version of MTX, was synthesized and tested for its ability to induce dimerization of DHFR. Gel-filtration analysis of purified DHFR confirmed that, in vitro, the protein was a monomer in the absence of dimerizer drug; in the presence of bisMTX, a complex of twice the monomeric molecular weight was observed. Furthermore, the off-rate was found to be 0.0002 s −1, ∼100 times slower than that reported for DHFR–MTX . Interestingly, the addition of excess bisMTX did not result in formation of the binary complex (1 protein:1 dimerizer) over the ternary complex (2 proteins:1 dimerizer), which suggests cooperative binding interactions (affinity modulation) between the two DHFR molecules in the bisMTX:DHFR 2 ternary complex. Conclusions: The combination of DHFR and bisMTX provides a new CID system with properties that could be useful for applications in vivo. Formation of the bisMTX:DHFR 2 ternary complex in vitro is promoted over a wide range of dimerizer concentrations, consistent with the idea that formation of the ternary complex recruits energetically favorable interactions between the DHFR monomers in the complex.