We have identified a protein expressed by human and murine cells susceptible to coxsackievirus B3 (CVB3) infection and purified it from HeLa cells. This protein of approximately 45,000 Mr is expressed by HeLa cells and mouse fetal heart fibroblasts (susceptible to infection), and not by C3H murine fibroblasts or the human RD cell line (resistant). The protein was isolated from Triton X-100- deoxycholate lysates of HeLa cells by chromatography on concanavalin A-Sepharose, Affi-gel Blue, Phenyl Sepharose, and PBE94. The CVB3-binding fraction from PBE94 was blotted from SDS-polyacrylamide gel onto PVDF membrane for amino acid sequencing. Approximately 2 pmoles of CVB3-binding protein provided assignments for 26 consecutive residues: LSITTPEEMIEKAKGETAYLPXKFTL. This sequence corresponds neither to decay accelerating factor nor to nucleolin, both of which have previously been identified as CVB3-binding proteins, but does match two entries in GenBank. These data show that we have purified a novel CVB3-binding protein, the characteristics of which suggest the CVB group receptor has been purified. Identification of 26 amino acid residues in the protein and corresponding GenBank enteries will accelerate study of CVB tropism and the diseases caused by these viruses.