Intact female BALB/cCrgl mice, 3–4 weeks old, were pretreated with oestrogen and progesterone for 9 days. Whole mammary glands from these mice were cultivated for 5 days in a synthetic medium supplemented with aldosterone (A), prolactin (MH) and insulin (I), with and without thyroxine (T4) at concentrations ranging from 0·01 to 5 μg./ml.
A medium containing 1 μg. A +5 μg.MH +5 μg.I/ml. was generally optimal for lobulo-alveolar development. Addition of thyroxine to this combination resulted in a decrease in development which was highly significant at higher concentrations. However, when cultures were maintained in media containing suboptimal or low amounts of prolactin (1 μg. A + 3 μg. MH +5 μg. I/ml. and 1 μg. A + 1 μg. MH +5 μg. I/ml., respectively), the results indicate two possible effects of thyroxine: lower amounts of thyroxine had synergistic effects, whereas greater amounts had antagonistic effects on lobulo-alveolar development.