Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough analysis of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP(.)rapamycin(.)FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Analysis of the data shows that rapamycin binds to FRB with moderate affinity (K-d = 26 +/- 0.8 mu M). The FKBP12(.)rapamycin complex, however, binds to FRB 2000-fold more tightly (K-d = 12 +/- 0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiological conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.