RNA aptamers for tumor necrosis factor-alpha (TNF alpha), for which functionality was demonstrated in L929 cells, show only little affinity for the protein in vitro. Detailed investigation of the aptamer-protein interaction by surface plasmon resonance and quartz crystal microbalance analysis revealed that affinity is not the only crucial parameter for efficacy and functionality of those aptamers. Instead, the sensitive equilibrium of the monomeric and homotrimeric form of soluble TNF alpha decides on aptamer binding. Our results show that the field of application and the source of TNF alpha have to be carefully defined before selection of aptamer sequences.