The principles of fluorescence spectroscopy, and its utility in addressing biophysical problems will be reviewed. A key difference between fluorescence and most other optical spectroscopies is the time interval known as the fluorescence lifetime. This feature can be exploited in a variety of ways to examine molecular motion, which in turn can complement our understanding of structural assessments of macromolecular complexes. The inherent sensitivity of fluorescence detection also allows for single molecule detection limits to be realized, and spatial as well as time correlation techniques provide for diffusion and single particle tracking measurements to be made even in the complex intracellular environment. These latter studies have been empowered by exploiting advances made in molecular genetics that provide for specific intracellular labeling of macromolecules of interest.