The larval stage of Echinococcus multilocularis causes alveolar echinococcosis (AE) in various mammals, including humans. Traditionally metacestodes are maintained in the laboratory by serial transplantation passages into susceptible animals such as mice or gerbils. However, in animal models it has always been difficult to draw definite conclusions about the factors modulating metacestode differentiation, and investigations on gene expression and respective regulation have been hampered by the complexicity of the host-parasite interplay. This paper describes the maintenance and proliferation of E. multilocularis metacestodes as well as the formation of protoscolices in a chemically defined medium devoid of host influence. The interactive role of a heterologous human cell line (CACO2) in the in vitro development of metacestodes was also assessed. The morphology and ultrastructure of in vitro-generated metacestodes was studied using scanning (SEM) and transmission electron microscopy (TEM). Different cultivation procedures were analyzed in terms of expression of B- and T-cell epitopes and of the relevant laminated layer-antigen Em2; the exact localization of this antigen was further demonstrated by immunogold electron microscopy.