Although monoclonal in origin, mammary tumors acquire a marked heterogeneity of cell phenotypes, including a mixture of steroid hormone-sensitive cells and insensitive cells. We describe here long-term studies on the effects of androgen withdrawal on cloned androgen-responsive S115 mouse mammary tumor cells as a model system to investigate mechanisms by which tumor cells lose their steroid sensitivity. In the prolonged absence of androgen, the cells lost hormone-sensitive parameters reproducibly, including loss of proliferative response, saturation density response, cell morphology response, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)-related RNA. These experiments have demonstrated that when deprived of hormone in the long term, a clone of responsive cells gives rise reproducibly to a population of unresponsive cells in an ordered series of phenotypic changes. At the time when the cells lost all androgen response in terms of cell biology and MMTV-LTR-RNA, increased methylation of MMTV-LTR sequences in the DNA was detected. Thereafter recovery of androgen sensitivity has not been achieved in any of these parameters. The possible role of de novo DNA methylation in the progression to androgen autonomy of S115 cells is discussed.