Intracellular redox and energetic status play a crucial role in cardiovascular diseases and metabolic disorders. The physiological status of reducing agents, such as NADPH and NADH, and of high-energy molecules, such as ATP, is required for antioxidant system activity. For these reasons, an accurate measurement of adenine and pyridine nucleotides is fundamental. In this study we examined the preanalytical phase of reduced pyridine (RPN) and adenine and oxidized pyridine (AOPN) nucleotide assay in human whole blood. Different experimental conditions were applied to RPN alkaline and AOPN acid extracts to find the best analytical performance. Our results show that a good RPN and AOPN linearity (r from 0.994 to 0.999), recovery (near to 100%), and precision (coefficient of variation < 5%) were obtained when supernatant from acid and ultrafiltrate from alkaline extracts were neutralized, frozen, and thawed just before HPLC injection. Since NADH decays rapidly at -80 degrees C, RPN levels must be assayed within 72 h while AOPN can be stored for 1 month at the same temperature. An accurate and quantitative method for nucleotide determination can be obtained by applying the preanalytical conditions proposed in this study.