Neurofilaments (also called 10-nm filaments or intermediate filaments) from bovine brain were incubated with microtubule protein at 37 degrees C in the presence or absence of 1 mM ATP and in a buffer that allowed microtubule assembly. Falling-ball viscometry revealed that the (non-Newtonian) apparent viscosity of the ATP-containing mixtures is 5-20 times greater than that of the mixtures prepared without ATP. A larger ATP-dependent increase in viscosity (approximately 100-fold) was seen when purified tubulin replaced microtubule protein. The magnitude of the increase depended on the concentrations of both neurofilaments and tubulin. The presence of both neurofilaments and assembled microtubules was necessary for the increase to occur. The viscosity was drastically reduced by stirring or by cooling of the mixtures to 0 degrees C. Sedimentation velocity experiments, conducted at 35 degrees C on mixtures previously incubated at 35 degrees C, revealed the presence of a fraction of very rapidly sedimenting material (sedimentation coefficient greater than 1000 S) in the ATP-containing solutions but not in those prepared without ATP. It is concluded that an ATP-induced complex is formed between microtubules and neurofilaments. The observed complex may reflect interactions between microtubules and neurofilaments that are significant in vivo.