Chimeric proteins were produced using the green light–emitting luciferase of Phrixothrix vivianii (PxGr: λ_max = 548 nm) and the red light–emitting luciferase of Phrixothrix hirtus (PxRe: λ_max = 623 nm). Constructs containing residues 1–344 of the red light–emitting luciferase with residues 345–545 of the green light emitting one emitted red light (PxReGr; λ_max = 613 nm), while the reverse emitted green light (PxGrRe; λ_max = 552 nm). From these results we conclude that the region 1–344 determines the color of bioluminescence (BL) in railroad-worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light–emitting luciferase (PxGr) resulted in a ∼40 nm redshift on the BL spectrum (λ_max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red-emitting luciferases (from λ_max = 623 to λ_max = 600 nm) and in PxGr R215S (from λ_max = 585 to λ_max = 560 nm) mutant luciferase, but not in the green-emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.