The principles of chemiluminescence and the application of chemiluminescent labels and substrates in immunoassays are reviewed. Various immunoassay formats and all known classes of chemiluminescent molecules, including 1,2-dioxetanes, luminol and derivatives, acridinium esters, oxalate esters and firefly luciferins are described as well as the many sensitizers and fluorescent enhancers. Recent promising developments are discussed.