Laetispicine, which is a novel amide alkaloid isolated from the stem of Piper laetispicum, has been proven to possess antidepressant and antinociceptive effects. This study examined the pharmacokinetic characteristics of laetispicine in plasma and brain distribution in rats by a simple sensitive HPLCUV method. The separation was performed on a reverse-phase ODS column (250 mm × 4.6 mm, i.d., 5 μm) with a mobile phase composed of acetonitrile-water (75:25, v/v) as the mobile phase at a flow rate of 1.0 ml/min with UV detection at 259 nm. The calibration curve of laetispicine in rat plasma showed excellent linear behavior between 0.0055.0 μg/ml (r2 = 0.9992), and between 0.020.5 μg/ml (r2 = 0.9952) in rat brain samples. The lower limit of quantification (LLOQ) was found to be 0.005 μg/ml in rat plasma and 0.02 μg/ml in rat brain samples. This HPLC assay was a precise and reliable method for the analysis of laetispicine in pharmacokinetic studies. Laetispicine was rapidly and extensively transported across the blood-brain barrier (BBB) and distributed into different brain regions. © 2010 World Scientific Publishing Company & Institute for Advanced Research in Asian Science and Medicine.