In the presence of zinc, the protein tubulin assembles into two-dimensional sheets that are a useful model system for the study of both tubulin and microtubule structure. Tubulin sheets present an ideal protein structure for study with atomic force microscopy because they contain a two-dimensional crystalline protein lattice and retain many of the structural features of tubulin and microtubules. However, high-resolution imaging requires nonperturbative immobilization onto an appropriate imaging substrate. In this report, several substrates commonly used for scanning probe microscopy are evaluated for their ability to effectively immobilize tubulin sheets: mica, gold, highly ordered pyrolytic graphite, and carbon-coated electron microscopy grids. We hypothesize that the different intermolecular interactions presented by these substrates will affect the morphology of adsorbed tubulin sheets as well as the amount of other contaminating adsorbates. Tubulin sheets were successfully imaged on all of these substrates and structural characterization is reported. The most consistent results were obtained on carbon-coated electron microscopy grids, which preserved fine structural features of the sheets and had the least amount of contamination from the adsorption of unpolymerized tubulin. Images of tubulin sheets obtained with atomic force microscopy also compare favorably with published electron micrographs of sheets produced using similar procedures. This work demonstrates the importance of assessing substrate effects when studying two-dimensional protein crystals and identifies suitable substrates for immobilizing tubulin sheets.