Ezrin is a component of the microvilli of intestinal epithelial cells and serves as a major cytoplasmic substrate for certain protein-tyrosine kinases. We have cloned and sequenced a human ezrin cDNA and report here the entire protein sequence derived from the nucleotide sequence of the cDNA as well as from partial direct protein sequencing. The deduced protein sequence indicates that ezrin is a highly charged protein with an overall pI of 6.1 and a calculated molecular mass of 69,000. The cDNA clone was used to survey the distribution of the ezrin transcript, and the 3.2 kb ezrin mRNA was found to be expressed in the same tissues that are known to express the protein and at the same relative levels. Highest expression was found in intestine, kidney and lung. The cDNA clone hybridized to DNAs from widely divergent organisms indicating that its sequence is highly conserved throughout evolution. The amino acid sequence of ezrin revealed a high degree of similarity within its N-terminal domain to the erythrocyte cytoskeletal protein, band 4.1 and secondary structure predictions indicate that a second region of ezrin contains a long alpha-helix, a feature also common to band 4.1. The structural similarity of ezrin to band 4.1 suggests a mechanism for the observed localization to the membrane, and a role for ezrin in modulating the association of the cortical cytoskeleton with the plasma membrane.