Prior studies demonstrated that expression of the retinoblastoma (RB) protein in acute myelogenous leukemia (AML) is heterogeneous with low expression conferring a poor prognosis. The molecular change(s) responsible for low RB expression in AML are unknown. Since methylation of the RB promoter has been shown to result in decreased expression we hypothesized that this might explain some cases of low RB expression in AML. To investigate this hypothesis Southern blotting and PCR sequencing after bisulfite conversion were used to study the methylation status of the RB gene promoter. DNA and protein lysates were prepared from the mononuclear cell fraction from peripheral blood or bone marrow samples from 46 patients with newly diagnosed AML. By Western blot 16, 22 and 8 patients had low, elevated and hyperphosphorylated patterns of RB expression respectively using previously defined criteria. The SacI endonuclease cuts a 5.7-kb or 6.8 -kb fragment, depending on polymorphism, containing the RB promoter, detected by the probe p123M1.8 that covers the RB promoter region and exon 1. The methylation sensitive endonuclease SacII cuts twice within a key hairpin loop structure in the RB promoter that contains binding sites for AP1, Sp1 and RBF1. Others have demonstrated that methylation within this hairpin loop can decrease RB mRNA transcription by up to 92%. Comparison of the SacI and SacI + SacII digestion fragments showed no evidence of methylation in the promoter region of RB in any of the patients studied. DNA from the promoter region of 11 patients with no/low RB expression was subjected to bisulfite conversion and PCR sequencing. No evidence of methylation was seen by this method either. These results suggests that hypermethylation of the RB promoter region is at best an infrequent event in AML and that RB promoter hypermethylation is not the predominant cause of the low levels of RB expression observed in 20% of AML patients.