Cyclophosphamide, administered in doses of 20 and 40 mg/kg body weight to pregnant inbred CBA mice on the third day of gestation (60 h after the estimated time of ovulation) reduced the activity of non-specific alkaline phosphatase (E.C. 126.96.36.199.) in 84 h-old blastocysts in a dose-related fashion compared with controls (p < 0.02 in each case; comparison of groups by Kruskal Wallis analysis of variance). This effect was not demonstrated with 4 mg/kg body weight (p < 0.1). Forty mg/kg body weight, but not the lower doses of cyclophosphamide, significantly retarded the hatching of embryos from the zona pellucida, attachment to the culture dish, and the formation of trophoblast outgrowths when the blastocysts were subsequently cultured in vitro for 120 h. The growth of an expanded inner cell mass was impaired in the 20 and 40 mg/kg groups. The differentiation of the inner cell mass into endoderm and ectoderm was significantly affected in the 4 mg, 20 mg and 40 mg/kg groups (p < 0.05, p < 0.01 and p < 0.001 respectively). The possible relationships of these various findings are discussed in the text. These results suggest that alkaline phosphatase may be a useful indicator of impaired growth and differentiation of the inner cell mass after exposure of preimplantation embryos to teratogens.