The improved p-dimethylaminobenzaldehyde method for colorimetric determination of orotic acid was reported in the previous report. This paper describes the mechanism of the color development. It was elucidated that bromination of orotic acid results in the production of 5,5-dibromobarbituric acid and the brominated compound is reduced with ascorbic acid to barbituric acid which condenses with p-dimethylaminobenzaldehyde to yield an orange color, 5-(p-dimehytlaminobenzylidene)-barbituric acid.