We have previously isolated detergent-resistant membrane complexes from lysates of cultured kidney epithelial cells. These structures are enriched in proteins anchored by glycosylphosphatidylinositol, which are localized to the apical plasma membrane of epithelial cells, and have a distinctive lipid composition. We now report that the membranes contain p62yes, a non-receptor tyrosine kinase of the Src family. p62yes is the only major tyrosine-phosphorylated protein in the membrane complexes when they are isolated. Several proteins in the complexes can serve as substrates for phosphorylation by p62yes in vitro. The kinase is enriched at least 10-fold in the insoluble membranes compared to cellular membranes. p60c-src and p60v-src, which are highly homologous to p62yes, are not present in the complexes, even when the membranes are isolated from cells that overexpress the proteins. As p62yes is located near the apical plasma membrane of epithelial cells in several tissues in vivo, interaction of the kinase with other components of a detergent-resistant membrane microdomain may play an important role in transmembrane signal transduction.