The steroid hormone progesterone plays a central role in the reproductive events associated with pregnancy establishment and maintenance. Physiological effects of progesterone are mediated by interaction of the hormone with specific intracellular progesterone receptors (PRs) that are expressed as two protein isoforms, PR-A and PR-B. Both proteins arise from the same gene and are members of the nuclear receptor superfamily of transcription factors. Since these two isoforms were identified in the early 1970s, extensive controversy has existed regarding the selective contributions of the individual PR proteins to the physiological functions of progesterone. During the past decade, significant progress has been made in this regard using two complimentary approaches. First, analysis of the structural and functional relationships of each isoform using in vitro systems has generated compelling evidence to support the conclusion that PR-A and PR-B have different transcription activation properties when liganded to progesterone. Second, the advent of gene-targeting approaches to introduce subtle mutations into the mouse genome has facilitated the evaluation of the significance of observations made in vitro in a physiological context. Selective ablation of PR-A and PR-B proteins in mice using these technologies has allowed us to address the spatiotemporal expression and contribution of the individual PR isoforms to the pleiotropic reproductive activities of progesterone. Analysis of the phenotypic consequences of these mutations on female reproductive function has provided proof of concept that the distinct transcriptional responses to PR-A and PR-B observed in cell-based transactivation assays are, indeed, reflected in an ability of the individual isoforms to elicit distinct, physiological responses to progesterone. In PR-A knockout mice, in which the expression of the PR-A isoform is selectively ablated (PRAKO), the PR-B isoform functions in a tissue-specific manner to mediate a subset of the reproductive functions of PRs. Ablation of PR-A does not affect responses of the mammary gland or thymus to progesterone but instead results in severe abnormalities in ovarian and uterine function, leading to female infertility. These tissue-selective activities of PR-B are due to this isoform's ability to regulate a subset of progesterone-responsive target genes in reproductive tissues rather than to differences in its spatiotemporal expression relative to the PR-A isoform. More recent studies using PR-B knockout (PRBKO) mice have shown that ablation of PR-B does not affect ovarian, uterine, or thymic responses to progesterone but rather results in reduced mammary ductal morphogenesis. Thus, PR-A is both necessary and sufficient to elicit the progesterone-dependent reproductive responses necessary for female fertility, while PR-B is required to elicit normal proliferative responses of the mammary gland to progesterone. This chapter will summarize recent progress in our understanding of the selective contribution of the two PR isoforms to progesterone action.