Iso-orotic acid was found to be very pH sensitive in water and deuterium oxide. Identical spectral curves were obtained in both solvent systems. A lowering of the extinction coefficient at pH 6, as compared with pH 1, could indicate an ionization effect rather than enolization since no appreciable wavelength shift was observed. However, enolization is predominant in the region of pH 12. A confirmation that pK2 in iso-orotic acid refers to proton dissociation from an enolic species was obtained by determining the pK2 of iso-orotic acid in water and in deuterium oxide. In the presence of Cu(II) ions at pH 3, there was noted a slight shift towards the visible in the region of the uracil absorption band of iso-orotic acid. This slight shift is attributed to complexing through the ketonic oxygen and carboxyl group.