Cell lines established from donors with the inherited disorder ataxia-telangiectasia (A-T) exhibit exceptional sensitivity to ionizing radiation and chemicals known to produce increased levels of intracellular H2O2, suggesting a deficiency in glutathione-dependent detoxication reactions. Glutathione (GSH) biosynthesis in fibroblast and lymphoblast cultures derived from individuals known to be clinically unaffected, homozygous, or heterozygous for A-T was assessed. Following GSH depletion by diethylmaleate, fibroblasts (GM 3492) from a clinically unaffected individual resynthesized GSH at a rate approximately twice that observed in fibroblasts from known heterozygotes (GM 3488 and GM 3489). Unrelated A-T homozygote fibroblast lines GM 3487B and GM 5823 resynthesized GSH only very slowly. GM 3492 cells repleted intracellular GSH by 6 h after depletion, the heterozygote lines by 18 h. The A-T homozygotes replaced only 30% of the intracellular GSH pool by 24 h. A lymphoblast cell line from the A-T homozygote (GM 3189) also exhibited slow resynthesis after depletion. However, if these cells were permeabilized by treatment with digitonin, GSH synthesis proceeded at a rate exceeding synthesis in permeabilized or untreated normal lymphoblasts (GM 3323). The first enzyme in GSH synthesis, gamma-glutamylcysteine synthetase, was found to be elevated about 2.7-fold in A-T homozygote fibroblasts, suggesting that a substrate for GSH synthesis may be rate limiting. A-T homozygote lymphoblasts contained about 2-fold more gamma-cystathionase activity over other cell lines tested suggesting increased flux through the transsulfuration pathway for cysteine production in response to reduced cysteine supply. Transport of cysteine and cystine was found to be 8- and 5-fold slower in A-T homozygotes that did not affect fibroblasts while glutamate and methionine transport Vmax did not differ among the cell lines tested. These experiments demonstrate that cells from A-T homozygotes are deficient in cysteine transport, thus limiting GSH resynthesis after a depleting challenge such as radiation or GSH-depleting xenobiotic compounds.