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To obtain drug resistance in newly established AML cell line KY821 with double minute chromosomes (DMs) and its subclone KY821A3 without DMs, they were cultured in increasing concentrations of methotrexate (MTX). KY821 became resistant against 2×10^M MTX (KY821/MTX), whereas KY821A3 did against 2×10^M MTX (KY821A3/MTX) in a same period. Enhanced enzyme activities of DHFR corresponded to increased DMs numbers and to amplifications of dihydrofolate reductase (DHFR) gene in both resistant clones. The amplified DHFR gene was proved to be located on DMs by in situ hybridization. These data indicated that the presence of DMs in KY821 would facilitate an aquisition of drug resistance. Unstable MTX resistance was shown to be associated with unstability of amplified DHFR gene located on DMs, and the massage (mRNA) of DHFR was less stable than gene amplification or cytogenetic alteration (DMs). In conclusion, KY821 is a very unique and valuable human leukemic cell line for studies of drug resistance and gene amplification.