Responses to environmental stresses in higher plants are controlled by a complex web of abscisic acid (ABA)-dependent and independent signaling pathways. To perform genetic screens for identification of novel Arabidopsis (Arabidopsis thaliana) loci involved in the control of abiotic stress responses, a complementary DNA (cDNA) expression library was created in a Gateway version of estradiol-inducible XVE binary vector (controlled cDNA overexpression system [COS]). The COS system was tested in three genetic screens by selecting for ABA insensitivity, salt tolerance, and activation of a stress-responsive ADH1-LUC (alcohol dehydrogenase-luciferase) reporter gene. Twenty-seven cDNAs conferring dominant, estradiol-dependent stress tolerance phenotype, were identified by polymerase chain reaction amplification and sequence analysis. Several cDNAs were recloned into the XVE vector and transformed recurrently into Arabidopsis, to confirm that the observed conditional phenotypes were due to their estradiol-dependent expression. Characterization of a cDNA conferring insensitivity to ABA in germination assays has identified the coding region of heat shock protein HSP17.6A suggesting its implication in ABA signal transduction. Screening for enhanced salt tolerance in germination and seedling growth assays revealed that estradiol-controlled overexpression of a 2-alkenal reductase cDNA confers considerable level of salt insensitivity. Screening for transcriptional activation of stress- and ABA-inducible ADH1-LUC reporter gene has identified the ERF/AP2-type transcription factor RAP2.12, which sustained high-level ADH1-LUC bioluminescence, enhanced ADH1 transcription rate, and increased ADH enzyme activity in the presence of estradiol. These data illustrate that application of the COS cDNA expression library provides an efficient strategy for genetic identification and characterization of novel regulators of abiotic stress responses.