The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E(1) (PGE(1)) and by fluoride was observed. The responsiveness to PGE(1) was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg(++) concentration was 10 mM; Mn(++) at 5 mM stimulated the enzyme but could not replace Mg(++) except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10(-4) M. The pH optimum was 7.5 and the Michaelis constant (K(m)) for ATP concentration approximated 10(-4) M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringer's solution at 0 degrees C, no loss of activity was observed at 1(1/2) and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0 degrees C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed.