Human trophoblast cells offer a unique in vitro model for the study of aspects of the dynamic processes occurring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro, the addition of cyclic AMP analogs, 8-bromo-cyclic-AMP or Sp-8-bromo-cyclic AMPS, promotes syncytiotrophoblast formation, as shown by the disappearance of immunostained E-cadherin and desmoplakin, and increased numbers of nuclei per syncytium. An antagonist of cyclic AMP, Rp-8-bromo-cyclic AMPS, and an inhibitor of the cyclic AMP-dependent protein kinase catalytic subunit, H-89, impair cell fusion. This led us to study the pattern of expression and subcellular localization of cyclic-AMP-dependent protein kinase subunits during syncytium formation. Cytotrophoblasts expressed the RIalpha and RIIalpha regulatory subunits and the Calpha and Cbeta catalytic subunits. RIalpha was down-regulated during syncytium formation. No change in RIIalpha protein levels was observed, but there was a drastic subcellular redistribution. RIIalpha located in the Golgi-centrosomal area of cytotrophoblasts was scattered throughout the cytoplasm of the syncytiotrophoblast. Interestingly, an accumulation of RIIalpha was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ. This suggests a key role of cyclic AMP-dependent protein kinase type IIalpha during cell fusion and microvilli formation, both of which are essential for the secretory and transfer functions of the syncytiotrophoblast.