The discovery of multicolored fluorescent proteins (FPs), in reef corals, that are close relatives of the green fluorescent protein (GFP) has led to what is now viewed as the second GFP revolution. Numerous GFP-type proteins, termed "reef FPs," have been cloned from reef organisms and many possess new colors, novel molecular characteristics, protein chemistry and many display unusual photophysical properties. Although some FPs have certain disadvantageous properties, such as the tendency to oligomerize or have slow maturation rates, reef FPs have been developed into versatile probes for cell biology and imaging applications. Screening of natural sources for novel GFP-type proteins continues to be valuable due to the need to expand the range of spectral colors, brightness, monomeric or dimeric states, faster maturation states, and photoactivity. Confocal imaging, coupled with microspectral detection, provides a rapid technique for in vivo characterization of FPs with desirable spectral and photoactive properties.