Phosphofructokinase interacts with both microtubules and microtubules containing microtubule-associated proteins to produce bundling and periodical cross-bridging of tubules. Immunoelectron microscopy using anti-phosphofructokinase antibodies provided direct evidence that the kinase molecules are responsible for the cross-bridging of microtubules. Limited proteolysis by subtilisin, a procedure that cleaves the N-terminal segment of the free enzyme as well as the C-terminal "tails" of tubulin subunits exposed on microtubules, showed that while phosphofructokinase becomes resistant, tubulin retains sensitivity against proteolysis within the heterologous complex. These data suggest that the N-terminal segment of the enzyme, but not the C-terminal "tail" of tubulin subunits, is involved in the interaction between the microtubule and the kinase. The phosphorylation of phosphofructokinase or microtubules containing microtubule-associated proteins by the cAMP-dependent protein kinase did not interfere with the heterologous complex formation. MgATP prevents phosphofructokinase binding to the microtubules, and it can displace the enzyme from the single microtubules. However, the bundled microtubules are apparently resistant to the MgATP dissociation effect. Modelling of the assembly process suggests that the tubulin-kinase complex is able to polymerize as the free tubulin. Vinblastine, an anti-mitotic agent, inhibits tubulin assembly; however, its inhibitory effect is partially suppressed in the presence of phosphofructokinase. Fluorescence anisotropy measurements indicated that kinase and vinblastine compete for tubulin binding with no evidence for ternary complex formation. This competitive mechanism and the ability of the tubulin-enzyme complex to polymerize into microtubules may result in the resistance of the tubulin-enzyme complex against the inhibition of assembly induced by vinblastine. Microtubules formed in the presence of vinblastine plus phosphofructokinase can be visualized by electron microscopy. A molecular model is suggested that summarizes the effects of MgATP and vinblastine on the multiple equilibria in the tubulin/microtubules/phosphofructokinase system.