Methylation of CpG sites in the promoter region can affect gene transcription. DNA derived from peripheral blood leukocytes (PBL) from the well-characterized clinical cohorts might be useful to study the influence of environmental factors on DNA methylation. However, these studies could be confounded by the heterogeneous nature of PBL. The aims of this study were to determine the impact of PBL distribution on methylation status of the androgen receptor (AR) promoter, and determine the associations between PBL distribution-adjusted methylation status of the AR promoter and AR-related phenotypes. PBL differential count analyses were performed at the time of blood sampling for DNA preparation in 170 elderly men. The DNA was bisulfite treated, and the methylation status of five CpG units in the AR promoter was analyzed using a high-throughput technique based on MALDI-TOF mass spectrometry. The degree of methylation of all the five investigated CpG units was strongly positively associated with the percent of lymphocytes in the PBL (r (s) = 0.17-0.49, P < 0.05). Furthermore, the PBL distribution-adjusted methylation status of a specific CpG unit in the AR promoter was significantly associated with body mass index (r (s) = 0.24) and other measures reflecting fat mass in elderly men. In conclusion, adjustment for PBL distribution needs to be done to be able to use DNA from whole blood for methylation analysis of the AR promoter and most likely also when investigating other promoters.