The cystine content of cystinotic fibroblasts increases with the length of time in cultivation. Four of five cystinotic cell strains showed an increase in cystine content with increased passage number. Cystinotic fibroblasts did not increase in cell number after 3 days in dialyzed fetal bovine serum. However, their cystine content increased almost 2-fold on a per cell number basis. Cystinotic cells incubated in cystine-free medium lost approximately 50% of their free cystine over a period of 12 hr and subsequently remained at about this level for 12 additional hr. Treatment of cystinotic cells with 1.0 mM dithiothreitol in complete medium reduced their free cystine content to about 25% of the initial level in 5 hr. The free cystine remained at this level after incubation overnight incystine-free medium without dithiothreitol. When dithiothreitol is added in the presence of a cystine-free medium, even more free cystine is removed from cystinotic cells (90%), and the free cystine content remains at this low level for as long as 48 hr if the cells are kept in a cystine-free medium. Cystinotic cells depleted of more than 50% of their intracellular free cystine by incubation in cystine-free medium reaccumulate cystine rapidly after replacement of cystine-containing medium. When cystinotic cells were depleted of their free cystine by treatment with dithiothreitol in a cystine-free medium and were then replaced in complete medium the intracellular free cystine reaccumulated rapidly, regaining its initial value in about 4 hr. The intracellular free cystine content found after 10 hr in complete medium reflected the cystine content of the medium. Cells in medium containing 0.266 mM cystine reaccumulated almost 60% more cystine than cells in medium with 0.066 mM cystine. When cystinotic and normal control fibroblasts were incubated for 2, 4, and 24 hr in either [35S]methionine or [35S]cystathionine, no radioactivity was recovered as cystine, cysteine, or glutathionine.