In the hormonally responsive 7,12-dimethylbenz(a)anthracene (DMBA)- or N-nitrosomethylurea (NMU)-induced mammary carcinomas, regulatory mechanisms have been altered such that these tumors retain their hormonal dependence for growth but possess only a limited ability to synthesize the mammary gland-specific milk proteins. Quantitation of casein mRNA levels revealed that very low levels of casein messenger RNA (mRNA) were expressed in both the DMBA- and NMU-induced tumors growing in virgin animals (0.1 to 0.4% of the maximally induced 8-day-lactating mammary gland). Growth of DMBA-induced tumors in pregnant rats and the treatment of NMU-induced tumor-bearing animals with thioproperazine indicated that the tumor casein mRNA levels were hormone inducible (3.4- and 2.1-fold for the DMBA- and NMU-induced tumors, respectively). However, casein mRNA levels were still only 1 to 2% of those found in the normal mammary gland under the same hormonal environment. Localization of the casein-synthesizing cells in the DMBA-induced tumors by peroxidase-antiperoxidase staining and a specific casein antiserum indicated that, in both control and hormone-treated tumors, the vast majority of cells (greater than 95%) were unstable to synthesize casein. The hormonal induction of casein mRNA sequences could be correlated with an increase in the number of cells synthesizing casein, which appeared as small clusters of cells throughout the tumors. Therefore, the loss of hormone-regulated differentiated function in these tumors, which maintained hormone-dependent growth, suggests the presence of a defective regulatory mechanism beyond the level of the hormone-receptor-complex.