Deregulated methylation of cytosine in DNA is a frequent finding in malignancy that is reflected by general genomic hypomethylation and regional hypermethylation that includes the myogenic gene Myf-3. In this study of 198 DNA samples from 186 patients with a wide range of lymphoproliferative disorders (LPD), the methylation status of Myf-3 was assessed to evaluate its significance in the diagnosis of malignant LPD. DNA was digested with the restriction endonucleases HpaII and MspI, and using the Southern blot (SB) technique, the size and density of fragments that hybridized with a Myf-3 probe were used to assign the methylation status. None of the samples from 45 patients from a wide age range with benign LPDs had evidence of altered Myf-3 methylation and there was no age-related methylation change. By contrast, 115/123 (93%) of samples from patients with non-Hodgkin lymphoma (NHL) or lymphoid leukemia had increased Myf-3 methylation. There was no methylation alteration in 22/24 (92%) of samples from patients with Hodgkin lymphoma (HL), nor in five of six samples from LPDs that had atypical histopathologic features which were not diagnostic of lymphoma, while the remaining sample of atypical LPD had hypermethylated Myf-3 fragments. There was an association between increasing Myf-3 methylation and higher histopathologic grade of malignancy within specific lymphoma categories. It is concluded that the detection of increased Myf-3 methylation is a sensitive and specific test of malignancy which may complement other molecular methods that are currently used for the assessment of clonality. It may be of particular diagnostic use in natural killer (NK) and null cell malignancies for which other indicators of clonality are lacking. Furthermore, methylation status may prove to be of potential prognostic value.