NS0 is a cholesterol-requiring mouse myeloma cell line widely used in the production of recombinant antibodies. We have previously reported that the deficiency of 17 beta-hydroxysteroid dehydrogenase type7 (Hsd17b7) is responsible for the cholesterol auxotrophy of NS0 cells. Here we demonstrate DNA methylation to be the mechanism underlying transcriptional suppression of Hsd17b7 in cholesterol dependent NSO cells. Analysis of the DNA methylation pattern revealed methylation of the CpG-rich region upstream of the Hsd17b7 transcription start site in NSO cells. This is in contrast to the unmethylated status of this sequence in a naturally isolated cholesterol independent revertant cell population (NSO_r). This transcriptional repression was relieved after treating cells with the demethylating drug, 5-azacytidine. Drug treatment also gave rise to high frequency cholesterol-independent variants. Characterization of revertants revealed substantially elevated transcript level of 17 beta-hydroxysteroid dehydrogenase type7 (Hsd17b7) gene along with hypomethylation of the CpG-rich region. These results affirm that deficiency of Hsd17b7 causes cholesterol dependence of NSO cells. Furthermore, induction of cholesterol independence by altering DNA methylation pattern alludes to the role of epigenetics in the metabolic adaptation of NSO cells. With the widespread use of NSO cells, this finding will have a significant impact on the optimization of recombinant antibody production processes. (c) 2005 Wiley Periodicals, Inc.