Bioluminescence imaging (BLI) allows detection of biological functions in genetically modified cells, bacteria, or animals expressing a luciferase (i.e., firefly, Renilla, or aequorin). Given the high sensitivity and minimal toxicity of BLI, in vivo studies on molecular events can be performed noninvasively in living rodents. To date, detection of bioluminescence in living animals has required long exposure times that are incompatible with studies on dynamic signaling pathways or nonanaesthetised freely moving animals. Here we develop an imaging system that allows: 1. bioluminescence to be recorded at a rate of 25 images/s using a third generation intensified charge-coupled device (CCD) camera running in a photon counting mode, and 2. coregistration of a video image from a second CCD camera under infrared lighting. The sensitivity of this instrument permits studies with subsecond temporal resolution in nonanaesthetized and unrestrained mice expressing firefly luciferase and imaging of calcium signaling in transgenic mice expressing green fluorescent protein (GFP) aequorin. This imaging system enables studies on signal transduction, tumor growth, gene expression, or infectious processes in nonanaesthetized and freely moving animals.