The effects of concentrate-to-forage ratio and buffer on rumen fermentation and production parameters were examined in four rumen-cannulated cows (240 +/- 18 d in milk) fed a total mixed ration ad libitum in a 4 x 4 Latin square design. The treatments were a 50:50 concentrate to forage ratio with [1.2% of dry matter, (DM)] and without (0% of DM) buffer and a 75:25 concentrate to forage ratio with (1.2% of DM) and without (0% of DM) buffer. Rumen pH declined in response to increased concentrate but was not influenced by buffer. In the absence of the buffer, rumen acetate declined and propionate was elevated at the higher level of concentrate inclusion. The milk fat concentration was lower for cows fed the high concentrate diet without buffer; however, the addition of buffer to the diet prevented the milk fat depression. Milk fat depression was associated with elevated trans-C18:1 fatty acids in milk, which provides additional support for an inhibitory effect of these fatty acids on mammary fat synthesis. We concluded that the potential of nutrition as a tool to alter milk composition is greater in later lactation as these animals are better able to cope with the negative effects of high grain diets, and the treatment response is greater than in early lactation.