N-Ethyl-2-pyrrolidone (NEP) is an industrial solvent that has been increasingly used to substitute N-methyl-2-pyrrolidone. NEP is under scrutiny in scientific and regulatory committees because of developmental toxic and teratogenic effects in rodents. The two postulated NEP metabolites 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI) have recently been detected in urine samples from the general population. Thus, the toxicokinetic characterization of these biomarkers of NEP exposure in humans is of relevance both in the occupational as well as the environmental field. We orally dosed 20.9 mg NEP to three male volunteers. These volunteers collected all their urine samples over a period of 4 days post dose. In these samples we identified and quantified the above postulated NEP metabolites 5-HNEP and 2-HESI and determined their urinary elimination kinetics and their metabolic conversion factors. After 4 days we recovered 50.7 % of the dose as these two metabolites in urine, 29.1 % as 5-HNEP and 21.6 % as 2-HESI. The largest share of 5-HNEP was excreted within 24 h post dose, while the major share of 2-HESI was excreted on day 2 post dose. We estimated an elimination half-time for 5-HNEP of approx. 7 h and for 2-HESI of approx. 22-27 h. While the elimination of 5-HNEP was basically finished 72 h post dose, significant amounts of 2-HESI were still eliminated after 96 h. Both biomarkers can now be used in human biomonitoring studies to extrapolate from urinary measurements to the NEP dose taken up and thus to evaluate the risk caused by exposure to this chemical.