In vivo permeability measurements of drugs in the colonic/rectal region in humans are difficult. A new instrument for the perfusion of a defined and closed segment in the colon/rectum was developed. The objective of this study was to evaluate its use for studying drug absorption mechanisms in the human rectum and to investigate the effect of transmucosal water absorption on drug permeability. Six healthy subjects participated at 2 separate occasions by using a modified system for segmental rectal perfusion. The system consisted of a multichannel tube with inflatable balloons and was endoscopically introduced into the rectum. The technique was considered acceptable by the following criteria; (a) high and reproducible recovery of PEG 4000, (b) stable residence time of the solution within the test segment, (c) flux of electrolytes that agrees with previous reports, (d) mass-balance absorption of antipyrine across the rectal barrier, (e) and good acceptability to the subjects. The permeability of antipyrine in the rectal region was increased by inducing net water absorption. D-glucose was not absorbed during any study periods. The present technique is valuable for studying drug absorption from the human rectum.