Expression cloning technology of cDNAs is a suitable tool for identifying novel functional properties of genes. Here, we generated a lentiviral cDNA library-expressing system for human T cells based on a site-specific recombination system of phage lambda for transferring cDNA libraries with a minimum loss of its complexity. The library-transduced CD4(+) T cells were challenged with wild-type human immunodeficiency virus type 1 (HIV-1), and the cells that acquired resistance to HIV-1-induced cytopathic effect (CPE) were selected. From these cells, CD14 was isolated and proved to inhibit the entry of HIV-1 and the HIV-1-induced CPE. This cloning system allows rapid identification of genes encoding novel properties in human T cells and probably other mammalian cells.