It is well established that prolactin (PRL) sustains, while prostaglandin F_2α (PGF_2α) curtails, progesterone production by the rat corpus luteum (CL). We have previously shown that the actions of both molecules converge on the 20α-HSD gene and control its expression in a dramatically opposed manner. In this investigation, we have found twelve more genes that are inversely regulated by PRL and PGF_2α. In addition to 20α-HSD, PGF_2α stimulated and PRL inhibited PGF_2α-receptor, phospholipase Cδ₁ and TGFβ₁ expression. In contrast PRL stimulated and PGF_2α inhibited the LH receptor, 11β-HSD2, sterol carrier protein 2, mitochondrial glutathione S-transferase (GST), GSTμ₂, inhibitory DNA-binding proteins 1, 2, and 3, and calcium binding protein 2. We have also identified new target genes for PRL and PGF_2α. PGF_2α stimulated the expression of genes involved in cell signaling such as cell adhesion kinase-β, ERK3, FRA2, IL-2 receptor, and 14-3-3 proteins. PGF_2α also up-regulated the expression of the sodium channel β₁, Na/K ATPase, annexin IV, GST7π, and P450 reductase. In contrast PGF_2α inhibited the expression of two genes involved in cell cycle: cyclin D2 and retinoblastoma related protein (Rb2/p130). It also inhibited genes involved in estradiol (P-450_AROM) and cholesterol biosynthesis (HMG-CoA synthase), as well as genes involved in tissue remodeling: VEGF and TIMP3. PRL had a profound inhibitory effect on the expression of genes encoding the ADP-ribosylation factor 3, annexin V and c-jun, yet increased the expression of P450scc, 3β-HSD, and SR-B1 (HDL-receptor), all genes involved in steroidogenesis. PRL also stimulated the expression of β₂-microglobulin, TIMP2, cytochrome c oxidase IV, cathepsin H and L, and copper-zinc superoxide dismutase as well as elongation factor SIII, heat shock protein-60 and mitochondrial ATP synthase-D. In conclusion, this investigation has revealed a “yin-yang” relationship between PRL and PGF_2α in regulating certain critical genes in the rodent CL, and has demonstrated novel regulation by these factors of other important genes involved in luteal function.