In plants, cortical microtubules (MTs) occur in characteristically parallel groups maintained up to one microtubule diameter apart by fine filamentous cross-bridges. However, none of the plant microtubule-associated proteins (MAPs) so far purified accounts for the observed separation between MTs in cells. We previously isolated from carrot cytoskeletons a MAP fraction including 120- and 65-kDa MAPs and have now separated the 65-kDa carrot MAP by sucrose density centrifugation. MAP65 does not induce tubulin polymerization but induces the formation of bundles of parallel MTs in a nucleotide-insensitive manner. The bundling effect is inhibited by porcine MAP2, but, unlike MAP2, MAP65 is heat-labile. In the electron microscope, MAP65 appears as filamentous cross-bridges, maintaining an intermicrotubule spacing of 25-30 nm. Microdensitometer-computer correlation analysis reveals that the cross-bridges are regularly spaced, showing a regular axial spacing that is compatible with a symmetrical helical superlattice for 13 protofilament MTs. Because MAP65 maintains in vitro the inter-MT spacing observed in plants and is shown to decorate cortical MTs, it is proposed that this MAP is important for the organization of the cortical array in vivo.