T he uptake of l-[³⁵S]cystine was studied in six cystinotic and six normal fibroblast lines grown for five days either on cover slips or in 32-oz plastic flasks. Cystinotics showed greater uptake than normals. The apparent K_t for cystine entry in both types of cells was 0.043 mM but cystinotic cells showed a higher maximum velocity of entry. A comparison of the fate of l-[³⁵S]cystine incubated for 20 min with monolayers of cells showed 30% and 15% of the intracellular ³⁵S to be l-cystine in cystinotic and normal cells, respectively. The ³⁵S effluxed more slowly from cystinotic than from normal cells after a 20-min preloading with l-[³⁵S]cystine. Identification of ³⁵S compounds in efflux media after 3 min showed 75% of the total ³⁵S was l-cystine with the remainder in cysteine and acidic sulfur metabolites of cystine with no essential difference between cystinotics and normals. In paired experiments, the specific activity of the effluxed l-[³⁵S]cystine after both efflux periods was the same as that entering the cell, thus indicating that the free l-[³⁵S]cystine had not exchanged with the pre-existing pool in the cystinotic cells. During 3 min efflux, the l-cystine pool in normal cells was depleted mainly by loss of free cystine. In cystinotic cells, a new steady state was attained after 21 min of efflux and the intracellular l-[³⁵S]cystine had the same percentage of total radioactivity seen after the initial 20-min uptake. After the rapid efflux of l-[³⁵S]cystine from normals, [³⁵S]cysteine and other labeled cystine metabolites appeared in the efflux media. By the end of a 3-min efflux, cystinotic cells had incorporated more label into reduced glutathione than had normal cells. However, when the new steady state was attained in cystinotics, the amounts of ³⁵S in glutathione were not markedly different in the two types of cells. Approximately 95% of the total label could be accounted for in free sulfur compounds.The data show an increased uptake and decreased efflux of cystine from cystinotic cells. However, it is not possible to conclude if these differences are due to primary changes in membrane function or to the reflection of metabolic defects without further investigation.