A modification of a cell isolation technique used in animal studies was developed to remove enterocytes from duodenal biopsy specimens. Citrate-ethylenediaminetetra-acetic acid treatment removed enterocytes from any underlying lamina propria and produced single cells and strips of cells. A mean (SEM) of 4.39 (2.06) x 10(6) cells was obtained from nine duodenal biopsy specimens. Enterocyte recovery was estimated enzymatically using alkaline phosphatase activity and was found to be 61%. Cytological assessment of the cells with CAM 5.2 showed that 98% of the cells isolated were enterocytes with an intact brush border. The cells responded well to vasoactive intestinal peptide stimulation in the absence of an exogenously added adenosine triphosphate regenerating system. The addition of vasoactive intestinal peptide to duodenal enterocytes produced a biphasic dose dependent increase in cyclic adenosine monophosphate production. Stimulation of these cells with 10(-13)M vasoactive intestinal peptide resulted in a 50% stimulation over basal value while 10(-6)M vasoactive intestinal peptide led to a fivefold increase in cyclic adenosine monophosphate production. We conclude that duodenal biopsy specimens are a good source of human intestinal cells for the study of enterocyte physiology. The cells were viable and highly responsive to vasoactive intestinal peptide.