The avian eggshell contributes to successful embryogenesis by acting as the major source of calcium. It has been suggested, moreover, that birds have evolved eggshells with different structures adapted to cope with different calcium requirements imposed by different growth rates and modes of development. Yet the mechanism of calcium removal from the avian eggshell by the embryo remains unknown. Using enzyme histochemistry, we here test an old hypothesis stating that calcium is released from the eggshell by means of acidification by the action of carbonic anhydrase (CA) in the extra-embryonic chorio-allantoic membrane (CAM). This enzyme catalyses the reversible reaction CO2+H2O <-> H+HCO3 and is known to take significant part in acid-base regulation in the body. We examined CAMs from two bird species, Coturnix japonica and Sturnus vulgaris, with very different growth rates and modes of development. The CAMs were examined for CA activity at the later stages of embryonic development. However, in all CAMs examined, staining for CA was absent, leading us to the conclusion that existing hypothesis about a link between CA and embryonic calcium mobilization from the avian eggshell is at present premature.