Imprinted genes are differentially methylated during gametogenesis to allow parent-of-origin-specific monoallelic expression. We previously demonstrated establishment of normal imprinting at four key imprinted genes in mouse metaphase II oocytes after in vitro follicle culture. Commercially available culture media feature a wide range of methyl donor levels. The aim of the present study was to examine the effect of low methyl donor (methionine, vitamin B12, folic acid, choline, and vitamin B6) levels during follicle culture on acquisition of DNA methylation at imprinted genes in mouse oocytes. Follicle culture performed under low methyl donor levels led to decreased antral follicle development (mean [SD] antral follicle rate, 87.5% [12.6%] vs. 97.7% [4.3%] in control conditions; P < 0.05) and to a dramatic decrease in polar body (PB) oocyte rate (mean [SD] PB oocyte rate, 38.7% [25.5%] vs. 96.1% [7.1%]; P < 0.001). The methylation status of differentially methylated regions (DMRs) of four key imprinted genes was studied (by bisulphite sequencing) in normal-looking PB and germinal vesicle breakdown-arrested oocytes obtained from follicle culture under low methyl donor levels. DMRs of Snrpn, Igf2r, and H19 showed no alteration in DNA methylation, but at Mest DMR in PB oocytes, we found a significant reduction in DNA methylation compared to that in control follicle culture (DNA methylation, 89.9% and 98.2%, respectively; P = 0.0014). In conclusion, restriction of methyl donors during follicle culture led to a dramatic decrease in PB oocyte rate but induced no or only minor DNA methylation alterations at the studied regulatory sequences of key imprinted genes in oocytes. © 2010 by the Society for the Study of Reproduction, Inc.