The avian eggshell primarily consists of calcium carbonate mineral (calcite) and matrix proteins. Here we review matrix-mineral relationships in the eggshell at the ultrastructural level using scanning and transmission electron microscopy, and describe the distribution of osteopontin (OPN) as determined by colloidal gold immunolabeling for OPN. A rich protein network integrated within the calcitic structure of the eggshell shows variable, region-specific organization that included layered fibrous planar sheets of matrix, thin filamentous threads, thin film-like surface coatings, vesicular structures and isolated proteins residing on cleaved {104} crystallographic faces of the eggshell calcite. Except for the vesicular structures, these matrix structures all immunolabeled strongly for OPN. Given the potent mineralization-inhibiting function of OPN, we discuss how this protein might regulate eggshell growth rate and inhibit calcification at specific compartmental boundaries to provide eggshell form.