The chicken eggshell supplies approximately 80% of the calcium found in the hatchling chick. The mobilization of eggshell calcium into the developing embryo involves the transepithelial transport of large amounts of calcium in a development-specific manner. The cells responsible for the transport of eggshell calcium into the embryonic circulation are the ectodermal cells of the chorioallantoic membrane. In this report, we present a method for the isolation and culture of chorioallantoic membrane ectodermal cells, which are amenable to direct experimental manipulation. Cell preparations are characterized with respect to the expression of an ectoderm-specific cell surface marker (transcalcin, a calcium-binding protein), and a specific enzymatic activity (elevated Ca(2+)-activated ATPase). Functional assessment of in vitro cellular calcium uptake by 45Ca2+ tracer kinetics indicates the persistence of a temperature-sensitive, rapid-influx pathway similar to that observed in vivo. The preparations of primary ectodermal cells present an in vitro system applicable to the experimental analysis of calcium metabolism and transport by the chick chorioallantoic membrane.