The controlled cDNA overexpression system (COS) was developed to identify novel regulatory genes in model plants as well as in other species that might have a particular valuable trait. The COS system (Papdi et al. Plant Physiol 147:528-542, 2008) is composed of a random cDNA library prepared in a T-DNA plant expression vector, under the control of the estradiol-inducible XVE promoter. Large-scale genetic transformation of Arabidopsis thaliana generates a transgenic plant population with randomly inserted cDNA clones. Overexpression of the inserted cDNA can create selectable phenotypes, allowing the facile identification and cloning of the responsible genes. Here we describe protocols to create and use the COS system for diverse purposes in plant biology.