Layer-by-layer (LbL) thin films containing insulin were prepared by alternately depositing insulin and poly(allylamine hydrochloride) (PAH) onto a solid surface at pH 7.4. The deposition behavior of the LbL films was studied using a quartz crystal microbalance, UV-vis absorption spectrometer, ζ-potential analyzer, and an atomic force microscope. The insulin-containing LbL films were stable in neutral pH media, but the films decomposed in solutions at pH 5.0 or lower. The decomposition of the LbL films can be rationalized on the basis of the loss of electrostatic interactions between insulin and PAH due to a shift in the net electric charge of insulin from negative to positive in acidic media. The films also decomposed to some extent in media at pH 9.0 and 10. A circular dichroism analysis showed that insulin retained its original conformation when released from LbL film into acidic solutions. © 2011 Elsevier B.V.