An in vitro preparation of Malpighian tubules was used to investigate the excretion of the polar cardiac glycoside, ouabain, in Oncopeltus fasciatus. Tubules were found to consist of at least two morphologically and physiologically distinct segments, both of which metabolized ouabain. The distal segment (segment II) secreted primary urine and ouabain. Secretion of ouabain by segment II was not observed to occur against a concentration gradient and increased with increasing fluid secretion. The proximal segment (segment I) reabsorbed fluid and ouabain but not metabolites. Ouabain was reabsorbed against a strong concentration gradient (23-fold), was independent of fluid reabsorption, and increased with increasing fluid secretion by segment II. In rapidly secreting Malpighian tubules (a situation of high cardiac glycoside secretion by segment II), the presence of segment I reduced the excretion of ouabain by 84-93%, mainly by reducing ouabain concentration. It appears excretory loss of cardiac glycosides can be reduced in O fasciatus and thus may be a factor in the sequestration of cardiac glycosides in this insect.