Six karyotypically distinct clonal cell lines isolated from each of two freshly resected human malignant gliomas were examined for heterogeneity of morphology, in vitro growth rate, and chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum (II). Each clone was identified karyotypically as having come from the parent tumor. The karyotypic deviations were primarily numerical; chromosome numbers ranged from hypodiploid to near-tetraploid. Three morphological types were recognized: astrocyte-like; squamous-like; and fibroblast-like. The growth rates differed among the clones; the doubling times ranged from 48 to 84 hr in those from one tumor and from 72 to 252 hr in the other. Chemosensitivity was measured by cytotoxicity and/or colony-forming assay. In both assays and in both tumors, heterogeneity of chemosensitivity response to both drugs was demonstrated among the different clones from the same tumor. Dose-response curves from some clones differed statistically (log-probit analysis) from those of others, and when the curves were parallel, their 50% effective doses often differed. For the cytotoxicity assay, the 50% effective doses of BCNU ranged from 43 to 94 microgram/ml and for cis-diamminedichloroplatinum (II), from 29 to 340 microgram/ml. For the colony-forming assay, the 50% effective doses of 1,3-bis(2-chloroethyl)-1-nitrosourea ranged from 4.5 to 7.0 microgram/ml and for cis-diamminedichloroplatinum (II), from 0.35 to 1.40 microgram/ml. No correlation was evident between the chromosome number, morphology, growth rates, or chemosensitivities of the clones. These results identified heterogeneity of chemosensitivity among cellular subpopulations in human malignant gliomas.