The luteinizing hormone-induced morphological and physiological reorganization of the bovine follicle is preceded by a profound and well-orchestrated modulation of gene expression. In the present study, the cell type-specific methylation profiles of CYP11A1, HSD3B1, and CYP19A1, genes that encode key enzymes of steroid hormone biosynthesis, were analyzed to elucidate whether epigenetic parameters such as DNA methylation might be involved in gene regulation during luteinization. Transcript abundance and DNA methylation levels were determined in granulosa and theca of large dominant and late preovulatory follicles and in large granulosa lutein cells isolated from corpora lutea cyclica and graviditatis. Levels of the steroid hormones progesterone and estradiol-17beta were monitored to assess the physiological status of individual follicles. From our results, we conclude that (1) individual, even closely neighboring, CpG dinucleotides can show very different methylation levels; (2) proximal (<300 base pair [bp] from the respective transcription start sites) but not distal CpGs show cell type-specific methylation levels; (3) higher methylation levels suggestively preclude high levels of gene expression; (4) DNA methylation is not involved in the transient (HSD3B1 and CYP11A1) respectively permanent (CYP19A1) down-regulation of gene expression in late preovulatory follicles; and (5) DNA methylation may have a role in the permanent shutdown of promoter 2-directed CYP19A1 expression in large (granulosa derived) lutein cells.