Monolayer and bilayer Langmuir-Blodgett films of poly(gamma-benzyl-L-glutamate) (PBLG) were prepared on highly oriented pyrolytic graphite (HOPG), mica, silicon, and glass. The films were imaged by atomic force microscopy (AFM) in the contact and tapping modes to develop imaging strategies for ultrathin polypeptide films and to assess the effect of substrate on film microstructure. A better understanding of the local polypeptide film structure could guide future methods of device fabrication. The AFM tapping mode enables long-term nondestructive imaging of PBLG mono- and bilayers for dimensions as small as 0.2 mum. In contrast, contact mode AFM readily damages PBLG films. PBLG has the best monolayer coverage on HOPG, although film is sometimes observed on each of the other three substrates. On HOPG, the monolayer has a predominantly "lacey" structure whereas for the hydrophilic substrates, mica, silicon, and glass, it is "banded" or "solid." HOPG rarely exhibits a continuous bilayer film whereas the other three substrates exhibit much better coverage as bilayers than as monolayers. The PBLG monolayer behaves as a banded template for bilayer formation in some areas of mica, and for all four substrates, the second layer appears to be oriented with respect to the first. The morphology and coverage of PBLG as a function of substrate is discussed in terms of hydrophilicity and forces of adhesion. Whether voids observed by AFM in the PBLG structures are present in the native film at the air-water interface of the Langmuir trough, or are induced by film transfer, remains undetermined.