Currently there is limited knowledge regarding the anatomy of the distal arrector pili (AP) muscle. A previous study implicated fibronectin and ⍺5β1 integrin binding as the anchor between the AP and the extracellular matrix (ECM). The purpose of this study was to strengthen this hypothesis. Serial frozen sections of human scalp skin were double-labeled via immunofluorescent staining for ⍺5β1 with fluorescein and fibronectin with rhodamine, followed by fluorescent microscopy. Granular staining for ⍺5β1 with fluorescein and smooth staining for fibronectin with rhodamine were seen at the periphery of the AP muscle bundles and along the distal fibers. Precise co-localization of ⍺5β1 and fibronectin was observed at the AP-ECM interface by means of a dual filter. Analysis of variance was used on the relative density of staining for each epitope. Staining for both epitopes was significantly brighter at the distal fibers than at the middle or proximal portions of the muscle. A computerized three-dimensional reconstruction provides a detailed picture of the microanatomy of the distal AP, which allows mathematical evaluation of the forces of contraction. The anatomic co-localization between ⍺5β1 and fibronectin strengthens our hypothesis that interaction of these epitopes mediates the attachment of the distal AP to the ECM. (J Am Acad Dermatol 2000;43:19-23.)