This investigation was concerned with factors which would maximize and inhibit L-cyst(e)ine uptake and glutathione synthesis of rat lenses cultured for 24 hours in a medium containing [35S]-L-cystine. The lenticular protein-free extract was separated and quantified by use of HPLC equipment which included an in-line radioisotope detector coupled with extensive post-run computer analysis.Six thiols were assessed for their ability to increase the uptake of L-cyst(e)ine and its utilization for glutathione synthesis. The most successful was 2-mercaptoethanol, which increased the L-cyst(e)ine uptake 3.6-fold and glutathione synthesis 2.9-fold. This work demonstrated that the rate of glutathione synthesis was directly proportional to the rate of uptake of L-cyst(e)ine.An inhibitor of glutathione synthesis, buthionine sulfoximine, in a concentration range of 0.11-3 mM, decreased uptake of the [35S]-label by one-third. The 3.0 mM concentration inhibited glutathione synthesis 98.7% and decreased glutathione 51% in 24 hours. The data indicate that half the glutathione disappeared in 23 hours in these cultured rat lenses. Because determination of this value did not depend upon the variable rate of cysteine transport, the value may approximate thein vivovalue.