A mouse preimplantation embryo culture system was utilized to characterize the in vitro embryotoxicity of petroleum creosote (PC), a complex mixture of aliphatic and polycyclic aromatic hydrocarbons. ICR mouse embryos, collected on d 3.5 of gestation (blastocyst stage), were exposed for 7 h to varying concentrations of petroleum creosote in serum‐supplemented culture medium. Parallel embryo cultures were exposed to PC in medium supplemented with rodent hepatic S9 micro‐somal fractions to monitor the role of bioactivation in PC‐induced embryotoxicity. Embryos were subsequently cultured in control medium for 72 h and observed for viability as well as specific, time‐dependent developmental end points—hatching and attachment to the culture dish at 48 h, and trophoblastic outgrowth with a distinct inner cell mass at 72 h. Embryonic viability varied in inverse proportion to PC concentration. Petroleum creosote caused embryolethal effects at concentrations of 33 μg/ml of culture medium and 54 μg/ml. Embryotoxicity was not observed at 22 μg/ml. Culture supplementation with rodent hepatic S9 fractions did not modify, either qualitatively or quantitatively, the embryotoxicity of PC in vitro. These findings implicate PC as a prenatal toxicant and support environmental and human health concerns regarding PC exposure from PC‐containing chemical waste sites.