Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA. These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the α subunit of the human IL-3 receptor (hIL-3Rα) by factor-dependent growth. CD2 (frequency, 1 in 104) and hIL-3Rα (frequency, 1 in 1.5 x 105) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.