The effects of nonmutagenic environmental exposures can sometimes be transmitted for several generations, suggesting transgenerational inheritance of induced epigenetic variation. Methyl donor supplementation of female mice during pregnancy induces CpG hypermethylation at the agouti viable yellow (A^vy) allele in A^vy/a offspring. Epigenetic inheritance occurs at A^vy; when passed through the female germ line, A^vy epigenotype is not completely "reset." We therefore tested whether diet-induced epigenetic alterations at A^vy are inherited transgenerationally. Female A^vy/a mice were weaned onto either control (n=6) or a methyl-supplemented diet (n=5). These F0 dams were mated with a/a males. All F1 and F2 A^vy/a females were weaned onto the same diet as their mothers, then mated with a/a males. F1, F2, and F3 A^vy/a offspring were classified for coat color, an indicator of A^vy methylation. In total, 62 F1, 98 F2, and 209 F3 A^vy/a mice were studied. As expected, average A^vy/a coat color was darker in the supplemented group (P<0.01). However, there was no cumulative effect of supplementation across successive generations. These results suggest that, in the female germ line, diet-induced A^vy hypermethylation occurs in the absence of additional epigenetic modifications that normally confer transgenerational epigenetic inheritance at the locus.--Waterland, R. A., Travisano, M., Tahiliani, K. G. Diet-induced hypermethylation at agouti viable yellow is not inherited transgenerationally through the female.