The relative developmental toxicity of four direct acting, alkylating agents was determined in primary cultures of differentiating rat embryo midbrain (CNS) and limb bud (LB) cells and compared with that observed in the rat whole embryo postimplantation culture system. The alkylating agents tested include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). These alkylating agents have been shown to produce developmental toxicity following either in vitro or in vivo exposure. Viability for both CNS and LB was assessed by a neutral red dye assay. Differentiation of CNS cells was assessed by hematoxylin staining of neurons; differentiation of LB cells was assessed by Alcian blue staining of extracellular proteoglycans. Relative potencies of these compounds in the cell culture system were not the same as those observed in the embryo culture system. Whereas rank order of potency in the cell culture system, for viability and differentiation, was MMS > MNU > ENU > EMS, rank order in the embryo culture system, for embryo lethality and malformations, was MNU > ENU > MMS > EMS. Effective concentrations for cell culture viability and differentiation by MNU and ENU in cell culture were about three to nine times higher than comparable values previously reported for embryos, while effective concentrations for MMS and EMS were two to seven times lower than those observed in the embryos. Differences in potency between the two culture systems may be related to differences in formation and repair of DNA adducts, as well as differences in culture conditions.