The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2a synthesis following PGF2a treatment at either early-(day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P!0.01) 4-to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P!0.01) COX-2 and PGE2-9-K basal activities, and PGF2a synthesis rate, but higher (P!0.01) PGE2 production. Independent of luteal stage, PGF2a treatment did not affect COX-1 activity. In day-4 CL, PGF2a induced an increase (P!0.01) in both COX-2 activity and PGF2a synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2a up-regulated (P!0.01) both COX-2 and PGE-9-K activities, and PGF2a production, but decreased (P!0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2a challenge and were more marked in day-9 CL. Our data suggest that PGF2a directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2a synthesis in an autoamplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2a in rabbits.