To identify genes whose expression is regulated by exogenous stimuli, we developed a gene trap strategy for high-throughput functional screening. We built a promoterless retroviral vector carrying the sequence of a green fluoresecnt protein–nitroreductase fusion protein downstream from a splice acceptor site. Fluorescence-activated cell sorting analysis of the transduced population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely the nitroreductase moiety allows pharmacological selection against constitutive green fluoresecnt protein–nitroreductase expression. Using hepatocyte growth factor stimulation of liver cells as a model, we combined stimulus administration with either positive or negative selection, to recover cells carrying traps in induced or suppressed genes, respectively. The two selection procedures yielded cell populations whose fluorescence consistently increased or diminished after ligand stimulation. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Responsive clones make a library of reporter cells in which the transcriptional control of the trapped genes can easily be further characterized. This selection procedure allows fast and efficient isolation and characterization of genes whose transcription is regulated by exogenous stimuli in a given cell line.