A study was made of the effects of pH and protic and aprotic solvents on the spectral properties of Renilla (sea pansy) luciferin and a number of its analogs. The results have made possible the assignment of two tautomeric forms of Renilla luciferin, one which absorbs maximally at 435 nm and another which exhibits an absorption maximum at 454 nm. Furthermore the results provide an explanation for the visible absorption characteristics of the photoproteins aequorin (lambda-max 454 nm) and mnemiopsin (lambda-max 435 nm). In addition a Renilla-like luciferin can be extracted from both of these photoproteins. This luciferin produces light with Renilla luciferase, at a rate dependent upon the concentration of dissolved oxygen, and in other respects is indistinguishable from Renilla luciferin in this bioluminescent reaction. The results suggest that the native chromophore in both photoproteins is Renilla luciferin (or a nearly identical derivative). The results also suggest that a hydroperoxide intermediate probably exists in photoproteins, on energetic grounds, and to account for the oxygen concentration independency of the rate of photoprotein reactions. This hydroperoxide may be attached initially to an amino-acid side chain (possibly indolyl-OOH, imidazoyl-OOH, or -SOOH) rather than to the luciferin chromophore.