In a previous paper (VanAerts et al., Toxicology in Vitro 1993, 7, 769-775) we have shown that the embryotoxicity of Cyclophosphamide was greatly enhanced when Cyclophosphamide had been added to a primary hepatocyte culture derived from Aroclor 1254-pretreated male rats (M(A)) and the medium from this culture was added to a post-implantation rat embryo culture. However, when medium from hepatocytes that had been derived from untreated male rats (M(C)), untreated pregnant rats (P(C)) or Aroclor 1254-pretreated pregnant rats (P(A)) was used embryotoxicity was low. We now present data on the concentrations of 4-ketocyclophosphamide, carboxyphosphamide and nornitrogen mustard in the primary hepatocyte culture media. 4-Ketocyclophosphamide was absent in media from P(C) or P(A) and present in low concentrations in media from M(C) and M(A). Carboxyphosphamide and nornitrogen mustard were present in all media, but their concentrations were several times higher in medium from M(A) than in media from other sources. This indicates that Aroclor 1254 pretreatment enhances Cyclophosphamide metabolism much more in male rats than in pregnant rats. It is suggested that both a greater inducibility of CYP2B1 and augmented stabilization of 4-hydroxycyclophosphamide by glutathione conjugation may be processes that contribute to the observed differences in metabolite concentrations.