The stimulant designer drug mephedrone is a derivative of cathinone - a monoamine alkaloid found in khat - and its effect resembles that of 3,4-Methylenedioxymethamphetamine (MDMA). Abuse of mephedrone has been documented since 2007; it was originally a 'legal high' drug, but it has now been banned in most Western countries. Using cDNA-expressed CYP enzymes and human liver microsomal preparations, we found that cytochrome P450 2D6 (CYP2D6) was the main responsible enzyme for the in vitro Phase I metabolism of mephedrone, with some minor contribution from other NAPDH-dependent enzymes. Hydroxytolyl-mephedrone and nor-mephedrone were formed in vitro, and the former was purified and identified by nuclear magnetic resonance (NMR). In four forensic traffic cases where mephedrone was detected, we identified hydroxytolyl-mephedrone and nor-mephedrone again; as well as 4-carboxy-dihydro-mephedrone, which has been previously described; and two new metabolites: dihydro-mephedrone and 4-carboxy-mephedrone. Fragmentation patterns for all detected compounds were determined by a UPLC-QTOF/MS(E) system, and a fragmentation pathway via a conjugated indole structure was proposed for most of the metabolites. Blood concentrations in the forensic traffic cases ranged from 1 to 51¿µg/kg for mephedrone, and from not detected to 9¿µg/kg for hydroxytolyl-mephedrone. In one case, urine concentrations were also determined to be 700¿µg/kg for mephedrone and 190¿µg/kg for hydroxytolyl-mephedrone. All compounds were detected or quantified with an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system and an ultra performance liquid chromatography-time of flight/mass spectrometry (UPLC-TOF/MS) system. Copyright © 2012 John Wiley & Sons, Ltd.