The cyclin D2 gene is not expressed in resting primary B lymphocytes or in group I Burkitt's lymphoma (BL) cell lines that retain the characteristics of authentic BL cells. Expression of cyclin D2 is induced in primary B lymphocytes following infection with Epstein-Barr virus (EBV) or transfection of the EBV genes EBNA-LP and EBNA-2. However, attempts to induce cyclin D2 expression in BL cell lines by the enforced expression of EBV genes were unsuccessful. Since the demethylation agent 5-azacytidine has been shown to modulate viral gene expression in BL cells, we explored the possibility that methylation plays a significant role in the control of cyclin D2 expression. We show that 5-azacytidine treatment of the Mutu CI 179 BL cell line led to expression of cyclin D2 RNA and that expression correlated with differences in the methylation status of a CCGG restriction enzyme site near the transcription initiation region of the cyclin D2 gene. Thus, methylation appears to play a direct role in the regulation of the cyclin D2 locus in BL.