The progesterone receptor (PR) is a ligand-dependent transcription factor that promotes progestin-stimulated expression of target genes. Two functional PR isoforms, PRA and PRB, are expressed in progestin-responsive cells. PRA and PRB have distinct roles in gene expression and in mammary gland development. One role of PRs in T47-D cells is regulating expression of vascular endothelial growth factor (VEGF), a potent angiogenic growth factor. This study explores the isoform specificity of this PR function using parental T47-Dco cells that express both PRA and PRB and clonal derivatives that express either PRA (YA cells) or PRB (YB cells) or lack PR (Y cells). Treatment with progesterone induces VEGF mRNA and protein approximately 2-fold in T47-Dco and YA cells and 3-7-fold in YB cells, suggesting that PRA inhibits PRB-dependent induction of VEGF. This is consistent with the observation that clinically relevant progestins induce a much higher level of VEGF in YB cells than in YA cells. Another novel finding in this report is that estradiol (10(-8) M) induces VEGF production from YB cells. However, this induction is not blocked by 100-fold excess tamoxifen or ICI-182,780. Moreover, both tamoxifen (10(-6) M) and ICI-182,780 (10(-6) M) function as agonists for VEGF in YB cells. Small interfering RNA against PR or estrogen receptor abrogated estradiol and tamoxifen induction, indicating that the agonist-like response of these compounds in YB cells is estrogen receptor and PR dependent. Estradiol, tamoxifen, and ICI-182780 also induce VEGF in BT-474 cells when their PRB levels were elevated by transfecting an expression plasmid for PRB, but not when the cells were transfected with vector alone. These results indicate that (a) PRB preferentially regulates VEGF expression in breast cancer cells and (b) PRB-enriched tumor cells may produce more VEGF, have a better developed vasculature, and potentially are more resistant to tamoxifen and ICI-182,780 than cells that express an equivalent or higher level of PRA than PRB. These results imply that PRB-enriched breast tumors may respond well to anticancer therapies that include inhibitors of angiogenesis.