BACKGROUND 5-Aza-2'-deoxycytidine (dAZA), causes hindlimb phocomelia in CD-1 mice. Studies in our laboratory have examined the hypothesis that compound- induced changes in gene expression may uniquely affect hindlimb pattern formation. The present study tests the hypothesis that dAZA causes limb dysplasia by inducing cytotoxicity among rapidly proliferating cells in the limb bud mesenchyme. METHODS Pregnant CD-1 mice were given a teratogenic dose of dAZA (i.p.) at different times on GD 10 and fetuses evaluated for skeletal development in both sets of limbs by standard methods. Using general histology and BrdU immunohistochemistry, limb mesenchymal cell death and cell proliferation were then assessed in embryos at various times post dosing, shortly after initial limb bud outgrowth. The effect of dAZA on early limb chondrogenesis was also studied using Northern analysis of scleraxis and Alcian blue staining of whole mount limb buds. RESULTS Compound related hindlimb defects were not restricted to a specific set of skeletal elements but consisted of a range of temporally related limb anomalies. Modest defects of the radius were observed as well. These results are consistent with a general insult to the limb mesenchyme. Mesenchymal cell death and reduced cell proliferation were also observed in both sets of limbs. The timing and location of these effects indicate a role for cytotoxicity in the etiology of dAZA induced limb defects. These effects also agree with the greater teratogenicity of dAZA in the hindlimb because they were more pronounced in that limb. The expression of scleraxis, a marker of early chondrogenesis, was reduced 12 hr after dAZA exposure, a time coincident with maximal cell death, as was the subsequent emergence of Alcian blue stained long bone anlagen. CONCLUSIONS These findings support the hypothesis that cytotoxic changes in the limb bud mesenchyme during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia after dAZA administration.