The differentiation of chorionic layers II and III and alterations of intercellular junctions within the developing placental labyrinth were investigated in rats. Between the 14th and 16th day of gestation, the trabecular structures of the labyrinth alter and increasing numbers of primary, secondary, and tertiary lamellae develop. Trophoblastic layer I remains multicellular. Layer III rearranges and cytoplasmic fusions result in a continuous cytoplasmic barrier intervening between the already syncytial trophoblastic layer II and fetal capillaries. Simultaneously, most of the “undifferentiated” cells within the labyrinth disappear. Thin sectioning anf freeze-fracturing reveal multiple images of intercellular junctions, particularly gap and tight junctions in the cellular and syncytial parts of layers II and III. Continuous and regularly arranged tight junctions occur as well as fragmented structures which are spaced irregularly. Gap junctions are occasionally found between the juxtasinusoidal regions of layers II and III on the 14th day. More often they occur within restricted areas near the center of the trabeculae, integrated within extensive tight junctional strands. Gap junctions increase in later stages of pregnancy and eventually become distributed between the interfaces of layers II and III in the primary lamellae.