Previous histochemical studies identified human placental lactogen (hPL) in the syncytial layer rather than in the cytotrophoblast cells of human placenta. However, these studies failed to rule out the possibility that de novo synthesis occurred in a cell type distinct from that in which the highest steady state level is accumulated after intercellular transport. TO address this point, localization of placental mRNA in trophoblasts was attempted by the technique of in situ hybridization. Term placental sections were incubated with [3H]cDNA transcribed from sucrose gradient-purified hPL mRNA. Slides were stained, fixed for autoradiography, and examined under the light microscope. The syncytial layer was covered with silver grains. The amount of this radioactivity was greatly reduced when placental fragments were incubated with globin [3H]cDNA or if hPL [3H]cDNA was hybridized to sections of human tonsil tissue. In addition, the amount of silver grains was greatly red uced when the sections were prehybridized with unlabeled PL cDNA and subsequently hybridized with labeled probe. These results suggest that hPL is synthesized in the syncytial layer of placental villi and that the appearance of hPL in this region was not the result of intercellular transport.