Actin monomers with bound ATP, ADP, or fluorescent analogues of these nucleotides exchange the nucleotide on a second time scale, whereas filaments assembled from each of these species exchange their nucleotide with the solution at least 1,000 times slower than monomers. Filaments assembled from either ATP-actin or ADP-actin are indistinguishable by electron microscopy after negative staining. The dynamic elasticity and viscosity of filaments assembled from ATP-actin or ADP-actin or mixtures of these two species are the same over a wide range of frequencies. These observations do not support a recent suggestion (Janmey, P. A., Hvidt, S., Oster, G. F., Lamb, J., Stossel, T. P., and Hartwig, J. H. (1990) Nature 347, 95-99) that ATP hydrolysis within actin filaments stiffens the polymer and alters both their structure and affinity for nucleotides. The difference in observations between these two studies may be related to time-dependent changes in ADP-actin prepared in slightly different ways.