Placental cytotrophoblast cells differentiate into the multi-nucleated syncytiotrophoblast and it has been suggested that this process is abnormal in placental tissue from pre-eclamptic/IUGR pregnancies [1]. Although there is an incomplete understanding of the differentiation process in normal cytotrophoblasts, it has been shown previously that EGF is necessary for efficient formation of syncytial trophoblasts in vitro[2]. We recently demonstrated that EGF activates the MAPK p38 and that this effect is independent of the growth factors ability to inhibit apoptosis [3]. In this study we hypothesized that inhibition of EGF induced p38 activation using two specific inhibitors, SB203580 and SB202190, would prevents EGF induced cytotrophoblast differentiation in vitro as judged by both β-HCG production and cell multi-nucleation. Cytotrophoblasts isolated from term human placentas were cultured with and without EGF (10ng/ml). EGF treated cells were also incubated with or without p38 inhibitors (SB203580 (10uM) and SB202190 (10uM)), in a controlled 5%CO2/20%O2 atmosphere for 5 days. Media was changed on days 1,3 and 5 of culture and β-HCG assayed. On day 5 cells were fixed with methanol:acetone and stained with anti-desmoplakin (cell membrane marker) and DAPI (nuclear marker) for immunoflourescence, prior to imaging and manual counting to determine the number of nuclei per trophoblast. Trophoblast like cells incubated with EGF demonstrated increased multi-nucleation as compared to control cell cultures (EGF 53%±2.1% versus control 14%±2% p<0.05, after 5 days of culture) in agreement with previously published data [2]. This effect was reversed by the addition of either p38 inhibitor to EGF containing cultures (EGF+SB203580 20%±1.1% versus 53%±2.1% p<0.05, EGF+SB202190 9%±0.4% versus 53%±2.1% p<0.05).β-HCG concentrations were also reduced in EGF treated cells with the addition of either SB203580 (β-HCG undetectable) or SB202190 (β-HCG reduced by 73%). Stimulation of p38 by EGF may be responsible for its pro-differentiation actions on cytotrophoblasts. This may be as a result of downstream signalling to cAMP response element [4], a known trophoblast differentiation stimulator in vitro, or an as yet unidentified downstream substrate of p38.