Lipophilic polymer membranes incorporating binding sites are widely used in various potentiometric, amperometric, and optical sensors. Here, we report on the biofunctional modification of the surface of a Ca2+-selective membrane. A photoactivatable biotin derivative was synthesized and covalently immobilized on a soft polyurethane membrane. The modified polymer was characterized by X-ray photoelectron spectroscopy (XPS) as well as by potentiometric measurements. The selective binding of streptavidin by the photo-cross-]linked biotin derivative was demonstrated. The surface coverage obtained with different experimental protocols was analyzed by autoradiography using [35S]-streptavidin. The new approach may significantly extend the scope of applicability of potentiometric sensors.