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The whole second thoracic mammary gland of estradiol-17β + progesterone primed 3- to 4-week-old BALB/c female mice was induced to pregnancy-like lobulo-alveolar morphogenesis after 6-day cultivation in a serum-free culture medium containing a "growth promoting" hormone mixture, insulin + prolactin + growth hormone (somatotropin) + estradiol + progesterone. No radioimmunologically detectable casein was present in these glands. Subsequent cultivation for another 6 days in a "lactogenic" medium with the hormones insulin + prolactin + cortisol produced abundant milk-like secretory material in the alveolar lumen. RNA of the mammary gland after estradiol + progesterone priming or cultivation in the "growth-promoting" medium failed to show a measurable amount of casein mRNA activity when assayed in a cell-free protein synthesis system derived from Ehrlich ascites ribosomes, rabbit reticulocyte factors, and tRNA. However, the glands sequentially cultivated in the "growth-promoting" and the "lactogenic" media showed a high level of casein mRNA activity in the heterologous cell-free protein synthesis system. Sodium dodecyl sulfate/polyacrylamide gel electrophoretic characteristics of the immunoprecipitable (by antibody to mouse milk casein) polypeptides directed by the mammary RNA induced in organ culture medium containing the lactbgenic hormones were similar to the characteristics of the polypeptides directed by mammary polysomes of lactating mice. These results demonstrate hormonal induction of a specific mRNA in a sequential two-step culture of an entire organ in a serum-free chemically defined medium.