Eggshell quality is a major concern to the poultry industry: eggs with poor-quality shells hatch poorly and are rejected in the processing plant. The eggshell gland (ESG) proteins and the matrix proteins, which participate in crystallization, fulfill important functions during formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We selected layers that consistently produced eggshells with specific abnormalities, and continued to do so after molting, and evaluated the expression of 2 genes-osteopontin (OPN) and calbindin-as related to particular eggshell abnormalities. These genes are synthesized by the ESG and appear to participate in the calcification process. When the ESG produces normal eggshells, OPN was expressed uniformly by all of the epithelial cells facing the lumen, and calbindin was expressed by the glandular epithelium. In contrast, in the layers producing pimpled eggs, OPN was expressed only in sections of the pseudostratified epithelium, separated by areas of cells devoid of OPN gene expression, whereas calbindin was expressed at much greater levels throughout the glandular epithelium. Almost no OPN gene expression was observed in the ESG of layers producing corrugated shells, but their pattern of calbindin expression was similar to but somewhat greater than that in ESG that produced normal eggshells. In cases in which eggs had cracks at the sharp or blunt poles, OPN was expressed only at the side opposite to the cracks, whereas calbindin was expressed at both sides equally independent of the cracks. The results suggest that synthesis of the proteins associated with the formation of eggshells with the various abnormalities is controlled by different mechanisms. This may imply that more than 1 strategy will be required to improve eggshell quality.