Morphological differentiation of a pluripotent cloned human embryonal carcinoma cell line, NEC 14, was induced with the addition of N,N'-hexamethylene bisacetamide (10(-2) M) for 3 days in vitro. Once differentiated, the NEC 14 cells temporarily lost both their proliferative capacity in vitro and their tumorigenic potential in the nude mouse. When culture was continued, the differentiated derivatives occasionally began to proliferate 4 weeks after the removal of the inducer, but the loss of tumorigenic potential was not recovered. Besides morphology and loss of proliferative and tumorigenic potential, the differentiated derivatives expressed both differentiation markers, HLA antigens, and intermediate filaments. This system is useful in the selection of differentiation modifiers in cancer therapy.