Adenosine (ADO) is an ubiquitous nucleoside with multiple physiological actions, but few procedures have been developed for its analysis in normal human plasma. This study describes an HPLC assay for determining ADO in normal human plasma: 2'-deoxycoformycin (2'-DCF) is added to inhibit adenosine deaminase and prevent ADO conversion to inosine, and a reversed-phase radial compression column is used for rapid separation of ADO from plasma ultrafiltrate. Results show quantitative ADO recovery, a limit of detection of 10 ng (2:1 signal-to-noise), good reproducibility (retention time SD = ±2%, peak height SD = ±2-3%), and linear standard curves in the range of 20-1000 ng ADO. Normal adult volunteer plasma ADO levels (with heparin to prevent clotting) are 135 ± 38 ng/ml, with no significant sex difference. Serum ADO levels are much higher (1512 ± 458 ng/ml) due to ATP breakdown during clotting, so plasma better represents true circulating ADO. The method is simple, rapid, reproducible, requires 2'-DCF to prevent ADO degradation, and can be applied to study ADO in different physiological and pathological conditions in man.