This study aims to determine the optimal dosing and administration route for azacitidine to reduce global DNA methylation levels in the peripheral blood of patients with hematologic malignancies. Seventeen patients were enrolled into one of five dose level treatment groups (3 at 25 mg/m², 4 at 50 mg/m², 4 at 75 mg/m², 3 at 100 mg/m², and 3 at 150 mg/m²) and received IV azacitidine at their respective dose on days 1-5 of cycle one. All patients received 75 mg/m²/day IV on days 1-5 of cycle 2. Subcutaneous dosing of 75 mg/m²/day on days 1-5 was used in cycle 3. Peripheral blood was collected on days 1, 3, and 5 of each cycle, and global DNA methylation was measured using bisulfite-PCR pyrosequencing of the DNA repetitive element LINE-1. 14 patients were evaluable for response with 2 CR, 2 PR, 7 SD and 3 PD reported. LINE-1 DNA methylation decreased by 1.37, 2.29, 4.81, 1.94 and 4.05% on day 5 for the 25 mg/m², 50 mg/m², 75 mg/m², 100mg/m² and 150 mg/m² cycle one dose levels respectively. Mean decrease in LINE-1 DNA methylation was 3.7% with 75 mg/m² IV and 3.4% by subcutaneous administration. The data indicates that 75 mg/m² azacitidine given IV or SC effectively leads to global DNA methylation reduction by LINE-1 analysis.