The effects of the differentiation inducing agents (DIAS), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF-7) were compared. All these agents reversibly caused a concentration-dependent growth inhibition in monolayers and markedly reduced colony-forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 microM), but cell replication ceased with NaBu (1 mM), forskolin (50 microM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in G0/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 microM)-treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 microM)-treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.