SUMMARY Transmucosal electrical potential difference and short-circuit current of an in-vitro preparation of mucosal epithelium of rat colon were almost abolished and active sodium transport ceased when ouabain (1 mmol/l) was present on the serosal side of the epithelium. A considerable fraction of the total ATPase activity in the mucosa was sodium dependent and concentrations of ouabain of 0·5 mmol/l or more completely eliminated the activity of this fraction. Adenosine triphosphatase activities in mucosa taken from normal, hypothyroid, aldosterone-treated and sodium-depleted rats were compared. The activity of the ouabain-sensitive, sodium-dependent fraction was similar in all groups except for the hypothyroid rats in which it was considerably reduced. There was no evidence from the present study that the increased active sodium transport by colonic mucosa, associated with aldosterone action or sodium depletion, depended upon a change in ATPase activity. In hypothyroidism, reduction of ouabain-sensitive ATPase activity may be responsible for the observed impairment of the sodium transport mechanism.