The syncytial trophoblast of the human placenta forms by the fusion of mononuclear cytotrophoblast cells. Cytotrophoblast cells only fuse with other trophoblastic cells, indicating a specificity to this interaction. To explore the cellular aggregation which precedes fusion, we examined the association of cytotrophoblast cells isolated from term placentae and JEG-3 choriocarcinoma cells, a cytotrophoblast-like cell line, in suspension culture. Cytotrophoblast cells were isolated by dispersion of chorionic villi in trypsin-DNase in Ca2+/Mg2(+)-free medium. JEG-3 cells were released from culture flasks by trypsinization in Versene-EDTA buffer. In suspension culture, each cell type aggregated forming tissue-like masses over a 24-hr period. Transmission electron microscope analysis demonstrated the formation of numerous desmosomes between the aggregated cells. In outgrowth culture, the aggregates created in suspension were maintained as microvilli-covered multicellular structures with hollow cores. The extent of aggregation was dependent upon the concentration of cells in the incubations with greater aggregation occurring with higher cell densities. Aggregation of both cytotrophoblast cells and JEG-3 cells progressed rapidly during the initial 10 hr of incubation and then continued at a slower rate. Aggregation took place in serum-containing and serum-free medium, but was impeded in Ca2+/Mg2(+)-free medium. Incubation of JEG-3 and cytotrophoblast cells in the presence of the protein synthesis inhibitor, cycloheximide, prevented aggregation, whereas the inhibitor of N-linked glycosylation, tunicamycin, did not. The inhibitor of RNA synthesis, actinomycin D, had no effect on the aggregation of the cells during the initial 6 hr of aggregation. These findings suggest that trypsin treatment in Ca2+/Mg2(+)-poor medium removed a protein(s) from the trophoblast cell surface which must be resynthesized for cell-cell association to take place.