Cyclic adenosine 3’,5’-monophosphate (cAMP) is an important 2nd messenger involved in signal transduction pathways in diverse organisms from bacteria to mammals. Extracellular stimuli, including variable ionic conditions and drugs, can modulate cytoplasmic adenylyl cyclase activity via their effect on transmembrane proteins such as ion channels and G-protein coupled receptors. Through its influence on downstream signaling pathways, cAMP modulates various biological processes including metabolism, differentiation, cell adhesion and neuronal signaling. Standard methods to determine intracellular cAMP levels include radioimmunoassay (RIA) and competitive enzyme-linked immunosorbant assay (ELISA). Although quite sensitive, these immunological methods are time consuming and entail cell lysis and extensive processing steps. In an effort to streamline the detection of intracellular cAMP in Tetrahymena thermophila, and to make it possible to measure fluctuations of cAMP in vivo and in real time, the GloSensor™ cAMP Assay (www.promega.com), designed for transient expression in mammalian cells, has been adapted for use in Tetrahymena. The assay is based on a chimeric protein that includes a cAMP binding domain fused to mutant forms of Photinus pyralis luciferase. Upon binding intracellular cAMP, conformational changes alter the chimeric reporter tertiary structure, resulting in a reconstituted luciferase activity and a large increase in light output in the presence of the GloSensor™ substrate. Tetrahymena t. strains expressing the GloSensor™ reporter from the constitutive BTU1 transcriptional promoter have been constructed. The stable transformant strains are able to take up the GloSensor™ substrate readily. Using 96-well plates (approx. 40,000 cell/well) and a plate reader with luminometer capabilities, cAMP levels can be measured to gauge the effect of various chemical compounds and growth conditions in a live-cell, non-lytic assay format. It is possible to monitor changes in cAMP levels in cells responding to stimuli in real time. This system will make it possible to screen complex compound libraries for ligands that affect G-protein coupled receptor (GPCR) function in Tetrahymena. An initial screen of the Pathogen Box (http://www.pathogenbox.org), composed of 400 diverse, drug-like molecules active against neglected diseases of interest, is currently ongoing.