The positioning of adhesion molecules relative to the microtopography of the cell surface has a significant influence on the molecule's availability to form adhesive contacts. Measurements of the ratio of fluorescence intensity per unit area in epi-fluorescence images versus total internal reflection fluorescence images provides a means to assess the relative accessibility for bond formation of different fluorescently labeled molecules in cells pressed against a flat substrate. Measurements of the four principal adhesion molecules on human neutrophils reveal that L-selectin has the highest ratio of total internal reflection fluorescence/epi intensity, and that P-selectin glycoprotein ligand-1 (PSGL-1) and the integrins alphaLbeta2 (LFA-1) and alphaMbeta2 (Mac-1) have ratios similar to each other but lower than for L-selectin. All of the ratios increased with increasing impingement, indicating an alteration of surface topography with increasing surface compression. These results are consistent with model predictions for molecules concentrated near the tips of microvilli in the case of L-selectin, and sequestered away from the microvillus tips in the case of LFA-1, Mac-1, and PSGL-1. The results confirm differences among adhesion molecules in their surface distribution and reveal how the availability of specific adhesion molecules is altered by mechanical compression of the surface in live cells.