CD28 (Tp44) is a human T-cell-specific homodimer surface protein that may participate in T-cell activation. We have isolated a cDNA clone encoding CD28 by a simple and highly efficient cloning strategy based on transient expression in COS cells. Central to this strategy is the use of an efficient method to prepare large plasmid cDNA libraries. The libraries are introduced into COS cells, where transient expression of surface antigen allows the isolation of cDNAs by way of monoclonal antibody binding. The CD28 cDNA encodes a highly glycosylated membrane protein with homology to the immunoglobulin superfamily and directs the production of a homodimer in transfected COS cells.