The aim of this study was to test the effect of actinomycin D on the expression of selected genes and to elucidate possible components of its apoptotic pathway in mouse embryos. Selected mRNAs and Trp53 protein were examined in blastocysts cultured for 24 h in vitro with or without the presence of a high concentration of actinomycin D. In all tested genes, the relative quantities of mRNA were significantly lower in treated blastocysts than in controls. The mRNA quantities of H2afz, Actb, Bax, Bad and Bcl2 were reduced at a similar rate, but the decreases in Bcl2l2 and Trp53 mRNA were significantly greater. Treatment with actinomycin D also changed the ratio between the mRNA levels of some pro-apoptotic and anti-apoptotic genes: the Bad/Bcl2l2 and the Bax/Bcl2l2 ratios were on average 4.39 and 2.66 times higher in the treated embryos than in the controls, respectively. Generally, treatment led to developmental arrest and significant increase in the incidence of cells with typical apoptotic features. However, its effect on Trp53 protein expression was not significant. The results suggest that mechanisms beyond the apoptotic effect of actinomycin D might include specific changes in the expression of pro-apoptotic and anti-apoptotic genes, shifting the expression ratio in favor of the pro-apoptotic ones. The results also show that the role of Trp53 is probably not crucial in this apoptotic pathway.