Elevated expression of tumour necrosis factor- (TNF-) is associated with adverse pregnancy outcome. This study has examined the expression of TNF- and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF- on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RT-PCR demonstrated TNF- mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF- expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF- (100ng/ml) for 72h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF--treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean SD 23.9010.42 vs 9.377.45, t-test, P0.0001) with no significant change in the numbers of trophoblast cells (19.978.14 vs 21.737.79, t-test, P=0.39). Within the trophoblast cell population, the TNF--treated outgrowths exhibited a significant increase in multinucleated cells (14.105.53 vs 6.375.80, t-test, P0.0001) and a corresponding significant decrease in mononucleated cells (5.873.60 vs 15.375.87, t-test, P0.0001). In summary, this study describes the expression of TNF- and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF- restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased expression of TNF- during trophoblast differentiation may be detrimental to pregnancy.