We investigated a specific relationship between the expression of annexin 5 and prolactin in the corpus luteum of pseudopregnant rats, with particular interest in gonadotropin-releasing hormone (GnRH) and apoptosis of luteal cells. The expression of ovarian annexin 5 mRNA was significantly decreased at mid-pseudopregnancy and recovered at the end, while it remained low on the corresponding day of pregnancy. The dopamine agonist CB-154, administered at mid-pseudopregnancy (Day 5), increased ovarian annexin 5 mRNA, while prolactin, given daily for three days to cycling rats, decreased it. An immunocytochemical study also showed that annexin 5 increased in the corpus luteum on days 6 and 7 of pseudopregnancy after treatment with CB-154 on day 5. The distribution of annexin 5-positive cells was not uniform in the corpus luteum and matched that of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells. Since GnRH stimulates annexin 5 mRNA expression in the gonadotropes, involvement of the GnRH receptor was examined. Local administration of a GnRH antagonist, Cetrorelix, to hemilateral ovarian bursa of pseudopregnant rats simultaneously receiving CB-154, abrogated both the expression of annexin 5 and the TUNEL reaction. The present results clearly demonstrate that prolactin decreases annexin 5 mRNA in the luteal cells during pseudopregnancy. Prolactin is suggested to suppress the local action of GnRH, which stimulates annexin 5 synthesis and apoptosis of functional luteal cells during pseudopregnancy.