Xenon-129 biosensors offer an attractive alternative to conventional MRI contrast agents due to the chemical shift sensitivity and large nuclear magnetic resonance signal of hyperpolarized Xe-129. Here we report the use of fluorescence spectroscopy and isothermal titration calorimetry (ITC) to determine xenon binding affinity and thermodynamics with a water-soluble triacid-cryptophane-A (1). 1 was synthesized in 10 steps with a 4% overall yield. Fluorescence spectroscopy measured an association constant of (1.7 +/- 0.2) x 10(4) M-1 in phosphate buffer at 293 K. ITC measurements at 293 and 310 K yielded association constants of (1.73 +/- 0.17) x 10(4) and (3.01 +/-0.26) x 10(4) M-1 and indicated a large entropic contribution to xenon binding in water. On the basis of these data, cryptophane 1 showed roughly 2-fold higher affinity for xenon than any previously measured compound. Remarkably, ITC measurements in human plasma at 310 K gave a similar binding constant, K-A = (2.19 +/- 0.22) x 10(4) M-1, which supports the development of Xe-129 NMR biosensors for biological applications.