We reported a procedure of serum anticholinergic activity (SAA) measurement and the reliability and reproducibility of the receptor binding assay, and we also described the usefulness of SAA measurement reflecting the anticholinergic activity (AA) in the central nervous system (CNS). According to the results of a 10 times repeated measurement of standard atropine binding, the relative error was between -5.5 and +3.7%, and we considered that measurement of SAA in our studies is accurate and validated. Downregulation of acetylcholine activates inflammation in both CNS and peripheral tissue, which causes AA in both sites. Therefore, changes of AA in the CNS link with SAA in the peripheral system even if a substance having AA does not penetrate through the blood-brain barrier. Then we redescribe issues that require attention in the measurement of SAA. It is generally defined that any SAA greater than the detection limit of a quantitative atropine equivalent level (≥1.95 n<smlcap>M</smlcap> in our study) is positive. According to previous studies, SAA is considered to be positive when its atropine equivalent is ≥1.95 n<smlcap>M</smlcap> and undetectable when this is <1.95 n<smlcap>M</smlcap>. Nevertheless, as a low SAA can act as AA in the CNS, we should assume that SAA might also be positive if its marker concentration is between 0 and 1.95 n<smlcap>M</smlcap>. In addition, SAA should be measured around 11 a.m. or somewhat later because of the diurnal rhythm of cortisol in humans.