Gradient distribution of bio-adhesive proteins can regulate multiple cellular processes, including adhesion, growth, and migration. The ability to control the cell function by changing the surface density of immobilized ligands has become increasingly important in design of implantable medical devices and tissue regenerating scaffolds. Recent techniques in fabrication of substrates with controlled surface properties allow the examination of cell sensitivity to a wide range of adhesion gradients. Understanding the mechanisms by which cells sense and respond to these directional cues warrants a quantitative assessment of macroscopic cellular response to the surface gradients, supported by predictive theoretical models. This article presents a theoretical basis to examine the effect of ligand gradients on cellular adhesion, using an equilibrium thermodynamic model. The model facilitates a systematic investigation of the complex interplay of cell–substrate specific adhesions, non-specific repulsions, and membrane elasticity. This purely mechanistic model predicts a biphasic dependence between the extent of cell spreading and its position across the gradient substrate. Biotechnol. Bioeng. 2010;105: 172–183. © 2009 Wiley Periodicals, Inc.