Significance Tau is a neuronal microtubule-associated protein linked to numerous neurodegenerative disorders, including Alzheimer’s disease. Several lines of evidence support tau aggregation as well as loss of its native interactions with microtubules as contributing to pathology. Here, we explored the largely overlooked first step of microtubule assembly, namely the interaction of tau with soluble tubulin heterodimers. Using single-molecule Förster Resonance Energy Transfer (smFRET), we determine the topological features of tau in complex with tubulin. Our results contrast differences in tau isoforms and underscore the importance of conformational flexibility in tau function.