The human cell surface antigen 4F2 is a disulfide-linked heterodimer consisting of a glycosylated heavy chain and a nonglycosylated light chain. The antigen is ubiquitously expressed on proliferating cells but only in resting cells from certain tissues. Its function has been proposed to relate to cellular Ca2+/Na+ exchange. We describe the molecular cloning of the 4F2 heavy chain gene and cDNA by a gene transfer approach. Part of the gene was isolated from a genomic lambda library constructed with DNA of a secondary transfectant L cell line that expresses 4F2 antigen. A gene-specific probe derived from the phage inserts was used to isolate two full length cDNA clones. Both cDNA clones directed the expression of 4F2 antigen in transfected mouse L cells. The 4F2 antigen heavy chain gene specifies a 2.1-kilobase mRNA with an open reading frame coding for a 529-residue protein of 58 kDa. The protein lacks an NH2-terminal signal peptide but contains an internal transmembrane-spanning region and four potential glycosylation sites in its COOH-terminal domain. We predict that the 4F2 antigen heavy chain is a transmembrane protein with a cytoplasmic NH2 terminus of 81 amino acids. The antigen shows no homology to known protein sequences.