Eisosomes are stable protein complexes at the plasma membrane, with punctate distributional patterns. Their formation and how their locations are determined remain unclear. The current study discovered that the formation and distribution of eisosomes are influenced by the cytoskeleton. Disassembly of either the F-actin or the microtubules leads to eisosome localization at hyphal tips of germinated macroconidia in Neurospora crassa, and treatment with a high concentration of the microtubule-inhibitor benomyl results in the production of filamentous eisosome patterns. The defect in the cytoskeleton caused by the disassembly of microtubules or F-actin leads to an increased formation of eisosomes. (c) 2021 Elsevier Inc. All rights reserved.