Although it is generally agreed that active sugar absorption in vitro is absolutely dependent on the presence of sodium ions on the luminal side of the mucosa, previous in vivo studies in the ileum of rat, dog and man have shown that active glucose absorption is almost as rapid from a sodium-free mannitol solution as from a sodium chloride solution. These experiments were performed in hopes of reconciling this discreptancy. Absorption of three actively transported sugars (glucose, galactose, and 3-O-methylglucose) having different apparent Km's, and of fructose (absorbed by a separate carrier-mediated process) were measured in the human ileum in vivo. The following observations were made: (1) Mannitol substitution for sodium results in only a slight reduction (23%) in the active absorption of glucose. (2) Magnesium substitution for sodium results in a greater depression (45%) of glucose absorption. (3) The apparent Km for glucose absorption is increased when sodium is replaced by magnesium, but the Vmax is not altered. (4) Magnesium does not depress glucose absorption or the apparent Km for glucose transport when sodium is present in the perfusing solution. (5) Neither sodium removal nor magnesium has any effect on fructose absorption. (6) Absorption of galactose and 3-O-methylglucose (low affinity sugars for the glucose carrier) is reduced by about 40 to 50% when mannitol replaces sodium, but magnesium substitution for mannitol in a sodium-free medium does not further depress absorption of these sugars. The following conclusions are suggested by these results: First, part of the discrepancy between previous in vitro and in vivo experiments was due to the type of test sugar (glucose versus glucose analogue) and the solute used to replace sodium in the luminal solution. Second, magnesium is more effective than mannitol in reducing sodium concentration at the glucose transport site on the brush border. Third, luminal sodium ions have an important effect on active sugar absorption in the human small intestine in vivo, as they do in vitro. And, fourth, there is a component of active sugar absorption (about one-half) which appears to be independent of luminal sodium ions in vivo.