The urinary concentrations of the main metabolites of methamphetamine (MA), specifically p-hydroxymethamphetamine-sulfate (p-OHMA-Sul) and p-hydroxymethamphetamine-glucuronide (p-OHMA-Glu), were directly measured in MA users and rats using an optimized LC-ESI MS method. The concentrations of the two conjugates in 50 MA human users' urine ranged from 0.09 to 88.6 microM (0.02-21.7 microg ml-1) for p-OHMA-Sul and from <0.05 to 7.13 microM (<0.02-2.43 microg ml-1) for p-OHMA-Glu; the ratios of sulfate to glucuronide (S/G ratios) ranged from 2.2 to 37.1 (13.8+/-8.1). The results demonstrate that the sulfation is quantitatively more important than glucuronidation for the conjugation of p-OHMA in humans. The urinary concentration time-dependency in two MA users also revealed that the conjugates were mostly excreted in urine within 3 days post-intake. In contrast, in rat, almost all of the conjugated p-OHMA (>99%) was excreted as the glucuronide in urine. These findings confirm that a large species variation exists in the conjugation of p-OHMA between humans and rats.