Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG-endoperoxide synthase (PTGS2), PGF2α synthase (PGFS) and PGE2 synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E2 and/or P4 on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up-regulated at the mid phase of pseudopregnancy. The effects of E2 and/or P4 treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up-regulated by E2 plus P4 at oestrus and the mid phase of pseudopregnancy and was also up-regulated by a single treatment with P4 at late pseudopregnancy (p < 0.05). Simultaneous incubation with E2 and P4 up-regulated PTGS2 gene expression at oestrus and mid-luteal phase (p < 0.05). Progesterone plus E2 significantly increased PGE2 secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E2 and/or P4 affected neither PGF2α secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up-regulated at the mid phase of pseudopregnancy. An increase in PGE2 secretion and up-regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E2 and P4 at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE2 are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.