During chemotherapy and regrowth of brain tumors, tumor-cell heterogeneity, and possibly tumor progression, may change as a result of both the selective forces and mutagenic effects of treatment. We have isolated and characterized drug-response variants of multicellular rat 9L brain-tumor spheroids exposed to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Ten colonies were isolated from spheroids disaggregated immediately after treatment, and 10 colonies were isolated from treated spheroids disaggregated after 1 week in suspension culture. The sensitivity to BCNU was determined by assays of sister chromatid exchange and colony-forming efficiency in monolayer cultures of each subline after a 1-hr exposure to graded doses of BCNU. Three classes of response were found: BCNU sensitivity increased, decreased, or was comparable to that of uncloned, parent 9L cells. Resistant phenotypes were predominant (8/10) in sublines from spheroids disaggregated immediately after treatment, whereas hypersensitive phenotypes (4/8) were isolated only from spheroids disaggregated after 1 week of regrowth. Since subpopulations isolated immediately after treatment do not have the same biological characteristics as those isolated after a period of regrowth, these data suggest that tumor-cell heterogeneity may be generated by distinct processes at various times during therapy. The predominance of hypersensitive sublines obtained by the regrowth protocol may have resulted from the recovery of cells that would have died if isolated but were instead able to repair the drug-induced damage when left in contact with neighboring, possibly resistant cells. Two resistant and two hypersensitive sublines were studied further.