Patterns of gene expression in normal and neoplastic rat mammary tissues were compared by cell-free translation of their total polyadenylated RNAs and by dot blot hybridization of the RNA to cloned complementary DNA probes for six of the major milk proteins, including: Mr 42,000 and 25,000 caseins, X-casein, whey phosphoproteins, kappa-proteins, and alpha-lactalbumin. Distinct but overlapping messenger RNA populations were evident from the translation patterns of normal virgin, pregnant, and lactating mammary glands. Dot blot analysis showed that each milk protein RNA had a different characteristic accumulation profile during pregnancy and lactation. The MTW9 and MCCLX mammary tumor lines, which are transplantable, prolactin dependent for growth, and produce alpha-lactalbumin, both showed high alpha-lactalbumin and Mr 42,000 casein messenger RNA activity. The tumors also had other milk protein RNA sequences, although in different proportions than at any stage during functional differentiation of normal adult mammary gland. Our results indicate that normal pregnant mammary gland expresses all of the abundant milk protein genes prior to detectable milk secretion. The patterns of gene expression in the two mammary tumors do not appear to correspond to any particular stage of functional differentiation of the normal mammary gland.