In 1973, a Cholinesterase Research Unit was established in Denmark (DCRU). The primary aim was to provide a central sendee for determining genotypes and activity of plasma cholinesterase (BChE) in patients showing abnormal response after succinylcholine. The purpose of the present study was, on the basis of 20 years experience with this Unit, to establish accurate reference intervals for BChE activity and inhibition values for the different genotypes of BChE. Also we wanted to evaluate the influence of age and sex on the BChE activity in genotypically normal patients. Plasma cholinesterase activity was measured using benzoylcholine as substrate. The genetic variations of the enzyme were identified using differential inhibitors, i.e: Dibucaine, Sodium Fluoride, Succinylcholine, Urea and Ro‐2–0683. We investigated 6,688 patients. The reference values for the 13 genotypes represented agree with previous findings. In genotypically normal patients, no age or sex differences were found in BChE activity in children below the age of 10 years. From the age of 10 years the activity decreased significantly in both males and females, the activity in females being significantly lower than in males. In females the activity was lowest in the age group 30–40 years, returning to prepuberty level at about 60 years of age. In males the activity decreased slightly up to 50–60 years of age. Hereafter the activity was stable or tended to increase slightly. Most genotypes could be recognized using the results of the different inhibition studies. We found the inhibitors Dibucaine, Sodium fluoride, Urea and Ro‐2–0683 most helpful, whereas succinylcholine was of less value. We conclude that though most genotypes can be recognized biochemically, several variants with heterozygous occurrence of an abnormal and a usual gene are still very difficult or impossible to differentiate in this way. This, together with the increasing number of possible genotypes, have increased the need for tests based on DNA analyses.