Photoactivatable fluorescent proteins bring new dimension to the analysis of protein dynamics in the cell. Protein tagged with a photoactivatable label can be visualized and tracked in a spatially and temporally defined manner. Here, we describe a basic rational design strategy to develop monomeric photoactivatable proteins using site-specific mutagenesis of common monomeric red-shifted fluorescent proteins. This strategy was applied to mRFP1, which was converted into probes that are photoactivated by either green or violet light. The latter photoactivatable variants, named PA-mRFP1s, exhibited a 70-fold increase of fluorescence intensity resulting from the photoconversion of a violet-light-absorbing precursor. Detailed characterization of PA-mRFP1s was performed with the purified proteins and the proteins expressed in mammalian cells where the photoactivatable properties were preserved. PA-mRFP1s were used as protein tags to study the intracellular dynamics of GTPase Rab5.