The color, brightness, and stability of a fluorescent protein are dictated by its amino acid sequence. Attempts to alter the physical properties of fluorescent proteins have focused on random amino acid changes directed at positions known to be a part of or near the fluorophore in the protein. In this study TagBFP and TagRFP, which differ by 13 amino acid mutations, are compared to understand which amino acids are critical for the fluorophore's color. Several site-directed mutants were created, and the in vivo fluorescence of the 17 different mutant proteins were evaluated. The fluorescence of the proteins was measured in vivo, skipping the time-consuming step of protein purification since the fluorescence was not affected by purification. Nearly all mutants exhibit blue fluorescence. The color conversion from blue to red required the mutation of a full set of six amino acids that contact the fluorophore.