Contrasted with partially hydrolyzed polyacrylamide (HPAM), the self-assembly behavior and microstructure of hydrophobically associating polyacrylamide (HAPAM) in aqueous solution have been studied by means of fluorescence spectrum, transmission electron microscopy (TEM), atomic force microscope (AFM), and apparent viscosity test in this article. The fluorescent probe analysis indicates that the HAPAM molecules will form associating aggregates in pure water. The results show that HAPAM can easily self-assemble to form an aggregate by hydrophobic driving force in dilute aqueous solution. The association of hydrophobic groups of the HAPAM causes the formation of supermolecule, so there are associating aggregates formed at very low concentration and then leads to the formation of network at a higher concentration. TEM and AFM measurements show that a distinct network structure has been formed in 1000 mg L(-1) of HAPAM solution, indicating the strong association of hydrophobic groups. These results are consistent with the viscosity measurement and reveal that the excellent viscosification of HAPAM is due to the association of hydrophobic groups in the aqueous solution. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci 123: 2397-2405, 2012