Biliverdin is an important pigment in the eggshell of chickens and other avian species. Determination of the biosynthesis site for biliverdin is essential for understanding the biochemical process and genetic basis of eggshell pigmentation. Either blood or the shell gland could be the biosynthesis site of eggshell biliverdin. A segregation population with full-sib sisters genotyped Oo and oo, which laid blue-shelled eggs and light brown eggs, respectively, was constructed in a native Chinese chicken breed. Ultraviolet spectrophotometry and HPLC were used to determine the biliverdin concentration in eggshells, blood, bile, excreta, and shell gland of both groups of chickens. Biliverdin content was significantly different between egg shells of blue-shelled and brown-shelled chickens (P < 0.01). Blood and bile were tested 3 to 4 h before oviposition, and excreta was tested randomly. Results showed no significant difference in biliverdin concentration in blood, bile, and excreta between the 2 groups. In the shell gland, the biliverdin contents for the blue-shelled and brown-shelled chickens were 8.25 +/- 2.55 and 1.29 +/- 0.12 nmol/g, respectively, which showed a significant difference (P < 0.01). Our results demonstrated that blood is not the biosynthesis site of the shell biliverdin. Biliverdin is most likely synthesized in the shell gland and then deposited onto the eggshell of chickens.