By a new procedure stable monolayer cultures were derived from spheroids in 8 out of 17 different human sarcomas (16 soft-tissue and I osteogenic sarcoma). Eleven of the sarcomas were obtained from patients undergoing surgery, and 6 from BALB/c nude mice carrying s.c. growing xenografts. The new procedure involves aggregation of single-cell suspensions into spheroids and cultivation of these in agar-coated flasks until the growth rate levels off, at which time the spheroids are transferred to uncoated flasks. Cells proliferating from the rim of adhering spheroids are trypsinized and aggregated to form new spheroids. By 3 to 5 such alternations, monolayer cultures were obtained that have now been subcultured for about 6 months. The cell lines all gave rise to colonies in a clonogenic soft-agar system, and upon s.c. injection into athymic nude mice 3 lines tested formed growing tumors. The histology of spheroids formed from late monolayer passages closely resembled that of the original tumors. That the new procedure is superior to other methods of establishing sarcoma cell lines is indicated by the fact that a stable monolayer culture could be obtained directly from the tumors in only 1/8 cases where the above procedure was successful, and in only 2 instances from soft-agar colonies derived from the tumors.