Inner cell masses (ICMs) were isolated by immunosurgery from giant blastocysts formed by the aggregation of three morulae. A layer of endoderm cells formed on the outer surface of these primary ICMs in vitro. When this layer was removed by immunosurgery, a secondary endoderm layer formed. Alphafetoprotein (AFP) was used as a biochemical marker to characterize visceral endoderm formation in these cultured ICMs. The immunoperoxidase reaction on sections of ICMs cultured for intervals up to 120 h in vitro showed that some primary endoderm cells contained AFP, but these were always in the minority. The secondary endoderm layer, on the other hand, was composed of predominantly AFP-positive cells. It is concluded that the primary endoderm contains mainly parietal endoderm cells, while the secondary layer contains visceral endoderm cells. A model is proposed for the consecutive differentiation of parietal and visceral endoderm cell types from the ICM of mouse blasto-cysts.