Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-I-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-l-ylalanyl-tRNA(CUA). Excimer formation (lambda(ex) 342 nm; lambda(em) 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.