Acetyl-L-carnitine (ALC), a naturally occurring endogenous compound, has been shown to improve the cognitive performance of patients with senile dementia Alzheimer's type, and to be involved in cholinergic neurotransmission. Because ALC is an endogenous compound, validation of the analytical methods of ALC in the biological fluids is very important and difficult. This study was presented validation and correction for plasma ALC concentrations and pharmacokinetics after oral administration of ALC to human volunteers. ALC concentrations in human plasma were corrected by subtracting the concentration of blank plasma from each sample. Precision and accuracy (bias %) for uncorrected ALC concentrations were below 2.6 and 6.5% for intra-days, and 4.0 and 9.4% for inter-days, respectively. Precision and accuracy (bias %) for corrected ALC concentrations were below 10.9 and 6.0% for intra-days, and 10.5 and 16.9% for inter-days, respectively. Quantitation limit was 0.1 microg/mL. After oral administration of a 500 mg ALC tablet to 8 healthy volunteers, the principle pharmacokinetic parameters were 4.2 h of the half-life (t(1/2,beta)), the area under the curve (AUC(0-8)) of 9.88 microg.h/mL, and 3.1 h of the time (Tmax) to reach Cmax. This study first describes the pharmacokinetic study after oral administration of a single dose of ALC in human volunteers.