Eleven human lymphoid cell lines, two T-cell lines, six B-cell lines and three non-T-/non-B-cell lines were evaluated for their asparagine dependence and for their chemotherapeutic susceptibility to asparaginase, cytosine arabinoside (ara-C), and 5-fluorouracil (FU). Two T-cell lines were asparagine dependent, whereas all B-cell and non-T-/non-B-cell lines were asparagine independent. These differences in nutritional requirements were consistent with as much as 5,000-fold differences in asparaginase sensitivity. B cells were found to be as much as 200-fold less sensitive to ara-C than T cells, irrespective of the benign or malignant nature of the cells or the presence or absence of EB virus infection. One non-T-/non-B-cell line with cell markers similar to the B-cell group behaved like a B-cell line. Two other non-T-/non-B-cells showed unique ara-C dose-response curves. FU sensitivity study revealed heterogeneity among B-cell groups. Non-T-/non-B-cell lines were uniformly FU insensitive. These differences in chemotherapeutic susceptibility were discussed in terms of usefulness as an in vitro model.