A primary cell culture from estrogen-withdrawn chicken oviduct has been developed in which the genes for the major egg white proteins, ovalbumin and conalbumin, are induced by steroid hormones. The responsiveness of the isolated cells depends on the combination of enzymes employed to dissociate the oviduct; trypsin and pronase are essential. Administration of estradiol to cultured cells is insufficient to induce ovalbumin mRNA (mRNAov) to levels achieved in vivo. Both insulin and a second steroid (a glucocorticoid, progestin, or androgen) are required for the induction of the ovalbumin gene by estradiol to the extent reached in vivo. Although low levels of a progestin or androgen can synergize with estradiol to obtain an induction of mRNAov, we have demonstrated that a physiological concentration of corticosterone (10-8 M) potentiates a large response to estradiol without causing an induction when added alone. When estradiol, corticosterone, and insulin are present in the culture medium, mRNAov increases from about 20 to 6500 molecules/cell by 55 h. With the same culture conditions, conalbumin mRNA increases from about 120 to 4300 molecules/cell by 52 h. After about 55 h, the level of egg white mRNAs decreases, and this reduction in mRNAov correlates with a diminished transcription of that gene. The data obtained with the cultured cells demonstrate that the induction of the ovalbumin gene requires the permissive effects of insulin and a second steroid (probably a glucocorticoid in vivo) to facilitate the response to estradiol.