Using spin hyperpolarized xenon (¹²⁹Xe) we investigate the impact of the local molecular environment on reversible host–guest interactions. We label Xe guest atoms that are temporarily bound to cryptophane-A hosts using the Hyper-CEST technique. By varying the length of the saturation pulse and utilizing an inverse Laplace transform we can determine depolarization times for the noble gas in different local environments, in this case biomembranes possessing different fluidity. We extend this technique to magnetic resonance imaging, mapping the spatial distribution of the different biomembranes. Such decays measured in biomembranes of 200 μM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were characterized by mono-exponential decays with time constants of τ_POPC = 3.00^+0.77_−0.61 s and τ_DPPC = 22.15^+5.19_−4.16 s. Analyzing both environments simultaneously yielded a bi-exponential decay. This approach may give further insights into saturation transfer dynamics of reversibly bound Xe with applications extending into biomedical diagnostics.