In an effort to find exploitable metabolic differences between human T and B lymphoblasts, we have compared the ability of lymphocytes of varying phenotype to grow in cystine-deficient medium. Only 6 of 12 human lymphoblastoid cell lines tested were able to utilize homocysteine thiolactone or cystathionine in place of cystine for growth. This difference in growth requirements was unrelated to the rate of cell division, the presence of Epstein-Barr viral genetic material, or whether or not the cell lines derived from benign or malignant tissues. Rather, all B lymphoblastoid cell lines grew in homocysteine thiolactone- or cystathionine-containing medium, while the T and non-T, non-B lymphoblastoid cell lines did not. Normal human peripheral blood T and B lymphoblasts did not respond to mitogens in the homocysteine thiolactone or cystathionine medium, but developed the ability to utilize these cysteine precursors after stimulation with concanavalin A, protein A, or Epstein-Barr virus. The differences in cysteine requirements among T and B cell lines may reflect a fundamental difference in de novo protein-synthesizing capacity of the two cell types.