An in vitro method is presented which measures valid, unidirectional uptake rates for lipids across the intestinal brush border. This method combines analysis by a newly devised, double isotope counting system for solubilized tissue with the use of a nonabsorbable marker to correct gross uptake determinations for contamination by adherent mucosal fluid. Of seven markers, only [(3)H]inulin measured adherent mucosal fluid volumes as much as 20% greater than the other markers. Diffusion of the nonabsorbable marker, as well as of the compound being studied, into the unstirred layer made the time course of uptake critically important. The time lag for diffusion of marker invalidates the use of 1-min incubation periods; however, a linear time course of uptake that intercepts essentially at zero was found for taurocholate and octanoate for periods of from 2 to 5 min. Working within this critical time period with jejunum, it was shown that tissue dry weight was an appropriate measure of the amount of tissue and that uptake rates for taurocholic, octanoic, and lauric acids were linear with respect to concentration. Tissue binding of compounds was not significant. The results demonstrate that careful use of the described method yields accurate measurement of unidirectional uptake rates of lipids across the brush border that are of critical importance in defining the characteristics of membrane penetration and the rate-limiting steps in fat and sterol absorption.