Epidermal growth factor (EGF) is thought to be important in normal mammary development. The presence of EGF receptors in breast cancer cells suggests that it may also have a role in regulating growth of tumors of the human breast. Using a complementary DNA probe for the human EGF precursor we have examined expression of this gene in a series of human breast cancer cells in long term culture. The T-47D cell line demonstrated the highest level of EGF mRNA. EGF expression was not detectable in the MCF-7, BT 20, or HBL 100 cell lines. Surprisingly, in both T-47D and ZR 75 cells, pretreatment with progestins which exert antiproliferative effects under the conditions used increased EGF mRNA levels approximately 6-fold above untreated controls. This effect, demonstrable with as little as 0.1 nM of medroxyprogesterone acetate, was apparent as early as 12 h after addition of progestin and was reversed with the antiprogestin RU 486. Dexamethasone, estradiol, and dihydrotestosterone had no effect on EGF expression in T-47D cells. There was no evidence that the increased levels of EGF mRNA were due to gene amplification. Immunoprecipitation of biosynthetically labeled T-47D conditioned medium with antibodies to human EGF and EGF-precursor revealed the presence of both Mr 40,000 and 18,000 products. Fully processed Mr 6,000 EGF was not detectable in either conditioned medium or cell lysate. These data provide unequivocal evidence for the expression of the EGF gene in some human breast cell lines.