AbstractFibronectin fibrils within the extracellular matrix play central roles in physiological and pathological processes, yet many structural details about their hierarchical and molecular assembly remain unknown. Here we combine site-specific protein labelling with single-molecule localization by stepwise photobleaching or direct stochastic optical reconstruction microscopy (dSTORM), and determine the relative positions of various labelled sites within native matrix fibrils. Single end-labelled fibronectin molecules in fibrils display an average end-to-end distance of ∼133 nm. Sampling of site-specific antibody epitopes along the thinnest fibrils (protofibrils) shows periodic punctate label patterns with ∼95 nm repeats and alternating N- and C-terminal regions. These measurements suggest an antiparallel 30–40 nm overlap between N-termini, suggesting that the first five type I modules bind type III modules of the adjacent molecule. Thicker fibres show random bundling of protofibrils without a well-defined line-up. This super-resolution microscopy approach can be applied to other fibrillar protein assemblies of unknown structure.