The chicken riboflavin carrier protein (RCP) is an estrogen induced egg yolk and white protein. Eggs from hens which have a splice mutation in RCP gene fail to hatch, indicating an absolute requirement of RCP for the transport of riboflavin to the oocyte. In order to understand the mechanism of regulation of this gene by estrogen, the chicken RCP gene including 1 kb of the 5′ flanking region has been isolated. Characterization of the gene structure shows that it contains six exons and five introns, including an intron in the 5′ untranslated region. Sequence analysis of the 5′ flanking region does not show the presence of any classical, palindromic estrogen response element (ERE). However, there are six half site ERE consensus elements. Four deletion constructs of the 5′ flanking region with varying number of ERE half sites were made in pGL3 basic vector upstream of the luciferase-coding region. Transient transfection of these RCP promoter deletion constructs into a chicken hepatoma cell line (LMH2A) showed 6–12-fold transcriptional induction by a stable estrogen analogue, moxesterol. This suggests that the RCP gene is induced by estrogen even in the absence of a classical ERE and the half sites of ERE in this promoter may be important for estrogen induction.