Interaction between porcine brain tubulin and neurofilaments was investigated with reference to network formation in vitro. When a mixture of tubulin and neurofilaments was incubated at 33--34 degrees C, its low-shear viscosity measured with a falling ball viscometer, was far above the sum of viscosities of the separate components and gelation was observed upon increasing the concentrations of both tubulin and neurofilaments above a certain level. Gelation that required Mg2+ and GTP was inhibited by Ca2+,Ca2+-calmodulin or ATP. Antimitotic drugs suppressed gelation at substoichiometric concentrations and cold treatment destroyed the formed gel, which indicated that gelation occurred in conjunction with tubulin polymerization. Assaying by centrifugation revealed that the amount of tubulin co-sedimented with neurofilaments was evidently larger in the presence of neurofilaments than in their absence. Furthermore, electron micrographs showed that a large number of microtubules which were shorter than usual were formed in the presence of neurofilaments. Interestingly, measurements with an Ostwald-type viscometer demonstrated that neurofilaments elevated the viscosity of the tubulin solution in a concentration-dependent fashion. In other words, neurofilaments had the ability to stimulate polymerization of tubulin. We conclude that polymerization of tubulin with neurofilaments produces three-dimensional networks in vitro.