Bioluminescence imaging has become a very popular tool for noninvasive monitoring of fundamental biological and molecular processes in small living subjects. Luciferases are light-emitting enzymes that can generate light (known as bioluminescence) after reacting with specific substrates. The emitted light is used as a detection system for luciferase activity, which acts as a "reporter" for the activity of any regulatory elements that control its expression. These enzymes are isolated from various organisms, conveniently modified for expression in mammalian cells, and are extensively used in molecular biology and cell culture experiments. Recent advances in optical technology have opened a new dimension for in vivo application of luciferase enzymes in biomedical research. The most commonly utilized luciferases for in vivo bioluminescence are isolated from two very different sources: firefly luciferase (or beetle luciferase) and renilla luciferase (isolated from sea pansy). Although both these luciferases can produce light following interaction with the substrates, structurally and biochemically they are very different. Here we describe the methods and applications of firefly and renilla luciferases in molecular imaging using small animals.