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Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192, 1993). A4 cDNA contains an open reading frame that predicts a polypeptide of 17 kDa. To determine the function of the A4 protein, we characterized its biochemical and physiological properties. Hydropathy analysis of deduced A4 amino acid sequence revealed four putative membrane-spanning alpha-helices. The hydrophobic nature of A4 was confirmed by its being extractable with organic solvents. Immunocytochemical studies of cells expressing A4 localized it to the endoplasmic reticulum. Moreover, A4 multimerized in vivo as determined by coimmunoprecipitation experiments. The four-transmembrane topology and biophysical characteristics of A4 suggest that it belongs to a family of integral membrane proteins called proteolipids, some of which multimerize to form ion channels. Subsequent electrophysiological studies of nuclei isolated from microinjected Xenopus laevis oocytes transiently expressing A4 showed the appearance of a 28-pS channel. Thus our studies indicate that A4 is a colonic epithelium-enriched protein localized to the endoplasmic reticulum and that, similar to other proteolipids, A4 multimerizes and exhibits characteristics of an ion channel.