The avian eggshell is a biocomposite ceramic consisting of minute amounts of organic matrix and a crystalline calcium carbonate (calcite) filler. It is formed by a well regulated spatio-temporal assembling process, where extracellular matrix proteins, especially the sulfated glycosaminoglycan anionic sites of specific proteoglycans, have been involved in nucleation and growth of the inorganic crystalline phase. Together with such extracellular matrix molecules, the activity of carbonic anhydrase, is crucial for the normal eggshell formation. Here, we studied the effect of dermatan sulfate and carbonic anhydrase on the in vitro calcification of nonmineralized eggshell niembrane-mammillae substrate at different pH and incubation times. Crystal morphology was analyzed by scanning electron microscopy. Crystal nucleation and growth was delayed at lower pH. Dermatan sulfate modified crystal morphology producing aggregates of large calcite crystals exhibiting a columnar morphology, contributing to the eggshell texture development. Carbonic anhydrase increased the velocity of crystal growth and eventually contributed to the fusion of the crystal aggregates to each other. Although, the effect of other macromolecules could not be ruled out, the combinatory effect of proteoglycans and carbonic anhydrase seems to be important for the control of eggshell formation. (C) 2004 Elsevier Inc. All rights reserved.