To define early molecular targets of progestin action, the differential display technique was used to identify genes with altered levels of expression in T-47D breast cancer cells treated with the synthetic progestin ORG 2058 for 3 h. PRG1 was first isolated as a 200-bp cDNA clone and its progestin regulation confirmed by Northern analysis. Cloning of the complete coding region of PRG1 revealed that it shared a high degree of amino acid sequence identity with isoforms of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from several tissues and species. Expression of PRG1 mRNA was observed in several normal breast epithelial and breast cancer cell lines and in a variety of human tissues, with highest expression in the breast, aorta, and brain. In T-47D cells, PRG1 mRNA was rapidly and transiently induced by progestins, expression peaking between 2 and 4 h and returning to control levels by 12 h. Progestin-induced increases in PRG1 mRNA were inhibited by the progestin antagonist RU 486 and occurred via the progesterone receptor. Progestin induction of PRG1 mRNA was also inhibited by actinomycin D but not by cycloheximide. PRG1 is therefore a novel human gene that is directly regulated by progestins via the progesterone receptor.