Proteorhodopsin is photoactive 7-transmembrane protein, which uses all-trans retinal as a chromophore. Proteorhodopsin subfamilies are spectrally tuned in accordance with the depth of habitat of the host organisms, numerous species of marine picoplankton. We try to find residues critical for the spectral tuning through the use of random PCR mutagenesis and endogenous retinal biosynthesis. We obtained 16 isolates with changed color by screening in Escherichia coli with internal retinal biosynthesis system containing genes for beta-carotene biosynthesis and retinal synthase. Some isolates contained multiple substitutions, which could be separated to give 20 single mutations influencing the spectral properties. The color-changing residues are distributed through the protein except for the helix A, and about a half of the mutations is localized on the helices C and D, implying their importance for color tuning. In the pumping form of the pigment, absorption maxima in 8 mutants are red-shifted and in 12 mutants are blue-shifted compared to the wild-type. The results of flash-photolysis showed that most of the low pumping activity mutants possess slower rates of M decay and O decay. These results suggest that the color-tuning residues are not restricted to the retinal binding pocket, in accord with a recent evolutionary analysis.