Total internal reflection microscopy was used to measure the total interaction between a 6 mu m glass sphere and a glass plate, separated by an aqueous solution containing 0.1-1.0 mM of KBr, when both surfaces are saturated with physisorbed polylysine. When the excess polylysine is completely removed from the solution, the sphere fluctuates around the secondary potential-energy minimum formed between double-layer repulsion and gravitational attraction. Subtracting gravity leaves a contribution from double-layer repulsion which decays exponentially with distance; the decay length is virtually identical to the Debye length calculated for each ionic strength. However, the presence of as little as 10 ppm of unadsorbed 26 kDa polylysine (rod length of 45 nm) causes a measurable attraction, although the most probable separation distance without polymer (150 nm) is much larger than the size of the macromolecule. Increases in the attraction with unadsorbed polymer concentration and decreases in the attraction with increasing KBr concentration correlate with the calculated osmotic pressure for two different molecular weights of polylysine, indicating that the attraction arises from the depletion of the polyelectrolyte from the gap between the sphere and the plate.