Intraduodenal lipid infusion stimulates phasic and tonic pyloric contractions and suppresses antral contractions. This study determined: (i) whether this response is sustained over 90 min; and (ii) the role of muscarinic mediation of this response. Antropyloroduodenal motility was recorded in 17 healthy volunteers with a sleeve/sidehole manometric assembly. Subjects received either a 90 min intraduodenal infusion of saline or triglyceride (20% Intralipid) at a rate of 1 mL/min; 30 min after the start of this infusion, the eight subjects who received triglyceride were given intravenous atropine 15 micrograms/kg over 30 s, followed by a maintenance infusion of 4 micrograms/kg/h until the study was completed. Intraduodenal triglyceride infusion stimulated isolated pyloric pressure waves consistently, producing a median rate of 2.4 per min after 30 min of triglyceride infusion, compared with a median rate of 0 per min pre-infusion. In intravenous saline studies, there was a reduction (P < 0.05) in the median rate of isolated pyloric pressure waves to 1.3 per min at 90 min, when compared with pyloric pressure waves at 30 min. Atropine reduced isolated pyloric pressure waves to a median rate of 0 per min, significantly different from preatropine (P < 0.01) and from intravenous saline studies (P < 0.0001). These results indicate that the phasic pyloric response to intraduodenal lipid persists during 90 min stimulation, albeit with significant attenuation. In humans, muscarinic cholinergic blockade prevents the occurrence of triglyceride stimulated localized pyloric contractions.