Many studies have implicated the cAMP Response Element Binding (CREB) protein signaling pathway in long-term memory. To identify small molecule enhancers of CREB activation of gene expression, we screened approximately 73,000 compounds, each at 7-15 concentrations in a quantitative high-throughput screening (qHTS) format, for activity in cells by assaying CREB mediated beta-lactamase reporter gene expression. We identified 1,800 compounds that potentiated CREB mediated gene expression, with potencies as low as 16 nM, comprising 96 structural series. Mechanisms of action were systematically determined, and compounds that affect phosphodiesterase 4, protein kinase A, and cAMP production were identified, as well as compounds that affect CREB signaling via apparently unidentified mechanisms. qHTS followed by interrogation of pathway targets is an efficient paradigm for lead generation for chemical genomics and drug development.