The Tg.AC transgenic mouse skin paint assay is one of the short-term carcinogenesis models that has been proposed as a replacement for 1 species in the conventional 2-yr bioassay required for safety testing of pharmaceuticals. In our initial efforts to evaluate the sensitivity and specificity of this model for human pharmaceuticals, 61% of the hemizygous Tg.AC mice in the positive control groups were refractory to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Tg.AC mice are reported to carry < or = 10 copies of a transgene consisting of a zeta-globin promoter fused to the v-Ha-ras structural gene with a terminal simian virus 40 (SV40) polyadenylation signal. Southern blot hybridization of genomic DNA from 66 tail biopsies using a zeta-globin probe revealed that all of the hemizygous. Tg.AC mice screened contained approximately 40 copies of the transgene and that mice unresponsive to TPA had lost a 2-kb BamHI fragment containing zeta-globin promoter sequence. By systematic screening of Tg.AC breeder mice for this diagnostic marker of phenotypic responsiveness, it should be possible to selectively enrich the Tg.AC mouse colony to consist exclusively of responders and to guard against further genetic instability.