AspCNU and SarCNU are two amino acid amide congeners (L-asparaginamide and sarcosinamide congeners) of chloroethylnitrosoureas. The in vitro myelotoxicity of these agents compared with BCNU at 1-8 micrograms/ml was determined in bone marrow cells from normal volunteers in the CFU-C assay. AspCNU and SarCNU were significantly (P less than 0.05) less myelotoxic than BCNU at equivalent microgram concentrations. SarCNU or AspCNU at 3 micrograms/ml demonstrate equivalent in vitro myelotoxicity to BCNU 1 microgram/ml. We used the human tumor stem cell assay (HTSCA) to investigate in vitro antitumor activity. We obtained four specimens of malignant glioma and one specimen of meningioma from patients not previously treated with chemotherapy. AspCNU and SarCNU were significantly (P less than 0.05) more active than BCNU at 1-3 micrograms/ml concentrations in the HTSCA in all four malignant glioma specimens. In the one meningioma specimen, BCNU was significantly (P less than 0.05) more active than either AspCNU or SarCNU at all concentrations studied. These results suggest that AspCNU or SarCNU at doses that should produce less myelotoxicity than BCNU may be more active than BCNU against gliomas.