The presence of amylose was demonstrated in hyphal walls of Neurospora crassa Shen and Dodge by silver proteinate staining for carbohydrates and differential digestion with β-amylase. The amylose was found to be localized in the innermost layer of the wall. Similarly, amylose-precipitating factor (APF) was also found to be localized in the same wall layer using ferritin-tagged antibodies to APF. Lipase-digestion of the isolated hyphal wall resulted in considerable reduction of the silver grain deposition in the innermost wall layer, suggesting the removal of amylose from this layer. Because lipid-free APF cannot bind to amylose, these observations indicate that APF and amylose in Neurospora are in a complexed form in the innermost layer of the hyphal wall as a structural component.