This report concerns the activity of choline acetyltransferase (ChAT), and the localization of ChAT and acetylcholine (ACh) in the human chorion during the 5th to 10th weeks of gestation. Radio-enzyme, immunohistochemical, and in situ hybridization assays were used. We found that ChAT activity increases as a function of gestational age between the 5th and the 9th weeks of pregnancy. At 5 weeks' gestation, ChAT was detected by immunohistochemical means mainly in cytotrophoblast and villous stromal cells, particularly in the cytotrophoblastic shell and cell columns. With the development of blood vessels at the 6th week, their endothelial cells also expressed ChAT. The amount of ChAT immunoreaction product deposits decreased in the villous cytotrophoblast by the 9th and 10th weeks when the layer of the cell became flat. By comparison, ChAT protein was detected only rarely in the syncytiotrophoblast layer during the gestation period studied. The ChAT gene transcript was demonstrated in most of the constitutive cells of the chorion. However, in contrast to the results of the immunohistochemical assays, positive hybridization signals for ChAT mRNA were evenly distributed over both cytotrophoblast and syncytiotrophoblast. On the other hand, the distribution of ACh immunoreaction products was almost coincident with that of ChAT.