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Artificial insemination (AI) of sows results in a significant elevation of prostaglandin F2α metabolite (PGFM) levels in peripheral plasma, whereas in mated sows such elevation is not seen. The aim of this study was to investigate whether boar seminal plasma (SP) has any effect on the release of PGFM, prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2) or interleukin-6 (IL-6) by in vitro cultured porcine endometrial (epithelial – pUE and stromal – pUS), cervical (pCE and pCS) and bovine endometrial epithelial cells (bUE). This study shows that boar SP inhibits the release of PGFM, PGF2α and PGE2 by porcine endometrial and cervical cells and bovine endometrial cells after 3 and 24 h incubation. Boar SP stimulated IL-6 release by pUE, pUS and even bUE after 3 h incubation. Tumour necrosis factor α (TNFα) stimulated the release of IL-6 by pUS only after 24 h incubation, but in the presence of boar SP, this stimulation was attenuated. The overall results from these in vitro studies give us possibility to understand the difference in prostaglandin response between mated and inseminated sows. Furthermore, we demonstrated that frozen-stored epithelial and stromal cells from pig endometrium, as well as from the cervix are suitable for studying the effect of SP on the release of prostaglandins. The only prerequisite is to incubate these thawed cells with arachidonic acid as a source for the synthesis of prostaglandins. A similar effect of boar SP on porcine and bUE cells may suggest inter-species reactivity.