This experiment determined the effects of infusion of mixtures of fat containing predominantly cis-C-18:1 or trans-C-18:1 fatty acids into the abomasum on responses of cows to glucose and norepinephrine challenges administered i.v. Six lactating Holstein cows, each with a rumen cannula, were arranged in two Latin squares with 21-d periods. The common basal diet contained 40% forage and 60% concentrate. Treatments were the uninfused control, 750 g/d of a cis fat mixture (65% high oleic sunflower oil and 35% cocoa butter), and 750 g/d of a trans fat mixture (93% shortening and 7% corn oil) infused into the abomasum via a tube that passed through the rumen cannula. Glucose challenges (0.4 mg/kg of BW, administered i.v.) were conducted on d 18, and norepinephrine challenges (0.7 mu g/kg of BW, administered i.v.) were conducted on d 19 of each experimental period. Despite a lower percentage of fat in milk for trans than for cis treatment, disappearance rates of glucose, secretion of insulin after glucose challenge, and appearance rates of NEFA and triglycerides after norepinephrine challenge mere similar between treatments. Thus, these data support the hypothesis that trans-C-18:1 fatty acids affect the synthesis of milk fat in the mammary gland of lactating cows