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In anesthetized dogs polyethylene catheters were inserted in the portal vein, hepatic vein, left femoral vein, and thoracic duct after which the pyloric region of the stomach was irrigated for 10 min with 1% (w/v) acetylcholine solution. Gastrin concentrations were measured by radioimmunoassay in venous sera and thoracic duct lymph prior to, during, and following acetylcholine irrigation. Gastrin concentrations were found to increase promptly in the portal vein sera, i.e., within 21/2 min of initiation of acetylcholine irrigation. Prompt increases in serum gastrin concentrations were also measured in hepatic and peripheral venous blood. Highest concentrations of gastrin were achieved in portal venous sera; the mean gastrin concentrations in hepatic vein sera were 60% of those in portal sera obtained simultaneously. Increases in peripheral venous gastrin concentrations were observed within 10 min of initiation of acetylcholine irrigation. Thoracic duct lymph gastrin levels were consistently less than peripheral venous serum gastrin levels in response to acetylcholine stimulation. These studies do not support the view that the thoracic duct lymph is a major route for conduction of immunoreactive gastrin to the peripheral circulation following acetylcholine stimulation of the stomach.