Interactions of the β-blocker drug, propranolol, with amphipathic systems have been studied using fluorescence spectroscopy. The results show a strong binding of propranolol with micelles of sodium dodecyl sulfate revealed through changes in the fluorescence spectrum and an increase in fluorescence lifetime. Quenching of propranolol fluorescence by iodide is used to demonstrate interaction with β-cyclodextrin. At high concentrations, self-quenching of propranolol fluorescence was also observed with κq=2.5×109 dm3 mol–1 s–1. Two-photon excited (630 nm) fluorescence lifetime imaging of propranolol in cells showed propranolol to be widely distributed in the cell cytoplasm, with fluorescence lifetimes shorter than in solution. The results suggest that intracellular propranolol is mainly confined within the aqueous cytoplasm and rather than membrane associated.