AimsThis study investigated the influence ofCES1variations, including the single nucleotide polymorphism (SNP) rs71647871 (G143E) and variation in copy number, on the pharmacokinetics of a single oral dose of 10 mg methylphenidate.MethodsCES1genotype was obtained from 200 healthy Danish Caucasian volunteers. Based on the genotype, 44 (19 males and 25 females) were invited to participate in an open, prospective trial involving six predefined genotypes: three groups with two, three and fourCES1copies, respectively; a group of carriers of theCES1143E allele; a group of individuals homozygous forCES1A1c(CES1VAR); and a group having threeCES1copies, in which the duplication,CES1A2, had increased transcriptional activity. Plasma concentrations of methylphenidate and its primary metabolites were determined at scheduled time points.ResultsMedian AUC ofd‐methylphenidate was significantly larger in the group carrying the 143E allele (53.3 ng ml−1 h−1, range 38.6–93.9) than in the control group (21.4 ng ml−1 h−1, range 15.7–34.9) (P < 0.0001). Median AUC ofd‐methylphenidate was significantly larger in the group with fourCES1copies (34.5 ng ml−1 h−1, range 21.3–62.8) than in the control group (P = 0.01) and the group with threeCES1copies (23.8 ng ml−1 h−1, range 15.3–32.0,P = 0.03). There was no difference between the groups with two and three copies ofCES1.ConclusionsThe 143E allele resulted in an increased AUC, suggesting a significantly decreased CES1 enzyme activity. Surprisingly, this was also the case in subjects with homozygous duplication ofCES1, perhaps reflecting an undiscovered mutation affecting the activity of the enzyme.