Hormone responsiveness of pregnancy-independent mammary tumors in SHN mice and pregnancy-dependent tumors in GR/A mice and in F1-hybrids between these strains was studied. Force-bred female mice with palpable mammary tumors were given subcutaneous injections of several hormones singly or in combination twice daily from 1 day before to 1 day after parturition. One group received a graft of three pituitary glands under the kidney capsule (3AP) during days 12-14 of pregnancy. One day after parturition, the in vivo incorporation of 3H-thymidine into DNA of normal and neoplastic mammary glands was determined as the index of DNA synthesis. Plasma prolactin in some groups of GR/A mice was assayed by radioimmunoassay. In GR/A mice, estradiol benzoate (EB: 0.5 mug X 2/day) or 3AP had no effect on either normal glands or tumors, despite an increase in plasma prolactin level. Progesterone (P:100 or 1,000 mug X 2/day) significantly increased DNA synthesis of both normal and neoplastic glands when compared to the controls, while the plasma prolactin level in this group was low. The administration of human placental lactogen (HPL: 100 mug X 2/day) stimulated DNA synthesis of normal glands only. P plus HPL promoted DNA synthesis of tumors more than P alone, but not P plus EB. While DNA synthesis of the tumors of SHN was never affected by these hormone treatments, the hormone responsiveness of tumors of F1-hybrids was almost the same as that of GR/A mice; the effect of P was prominent. Correlation of DNA synthesis between pregnancy-dependent mammary tumors and normal glands was significant only in groups treated with P alone or in combination with other hormones.