The photophysical properties of a fluorescently labeled amphiphilic polybetaine have been investigated by steady state and time-resolved fluorescence spectroscopy. The copolymer consists of N-isopropylacrylamide and N-phosphorylcholine-N'-ethylenedioxybis(ethyl)acrylamide units in similar to1/1 molar ratio, as well as 5 mol % of N-[(1-pyrenyl)-4-butyl]-N-n-(octadecyl)acrylamide. In water, individual copolymer chains associate in multichain aggregates held together by hydrophobic interactions between the hydrocarbon chains and by ion pair formation between the phosphorylcholine groups. By monitoring the changes in the ratio of the pyrene excimer emission intensity (I-E) to the pyrene monomer emission intensity (I-M), we established (1) that the polymer assemblies are disrupted by the addition of divalent salts, such as CaCl2 and (2) that interactions take place between the polymer and anionic, cationic, zwitterionic, or neutral surfactants. The mechanism of binding is discussed in terms of surfactant charge and chain length and compared to the association of surfactant to a copolymer of N-isopropylacrylamide and N-phosphorylcholine-N'-ethylenedioxybis(ethyl)acrylamide devoid of hydrophobic substituents.