Autocrine mediators released by the early embryo are implicated in the support of embryo development. Their mechanisms and timing of action, however, are uncertain. This study shows that their action is necessary during the 2-cell stage of development, at which time they do not act as classical growth factors, having no impact on the rate of cell-cycle progression. Rather, they act as survival factors, protecting embryos from subsequent cell death. Culture of zygotes for the first 48 h after insemination (but not the first 24 h only) at a high concentration of 1 embryo/μl before transfer to a concentration of 1 embryo/ 100 μl significantly improved development compared with culture at the lower concentration for 120 h. By contrast, if the initial 48 h of culture was at 1 embryo/100 Iμ, subsequent culture at 1 embryo/Iud caused no improvement. Platelet-activating factor (PAF) supplementation (0.186 μM) of media for the 48- h period after insemination (but not 24 h only) improved the development of embryos cultured at low embryo concentrations (1 embryo/10 μl) even when transferred to PAF-free media for the subsequent 72-h culture period. The ability of PAF to mimic the effects of higher embryo density implicates embryo-derived PAF as one of the autocrine mediators stimulating early embryo development. The cell death that occurred at low embryo concentration had a mixed phenotype: some cells had lost membrane integrity whereas others had intact membranes but punctate or fragmented chromatin. It is concluded that the action of autocrine diffusible factors, including PAF, are necessary during the 2-cell stage for subsequent survival and normal development of embryos to the blastocyst stage.