A randomized, two-way, crossover, bioequivalence study in 6 beagle dogs was conducted to compare the bioavailability of two peppermint oil formulations, soft capsule and hard capsule. The drug was given in a single dose of two capsules (total, 200 mg), and blood samples were withdrawn during the 12 h after drug administration. Menthol (CAS 2216-51-5) as the main component of peppermint oil was determined by a gas chromatography-tandem mass spectrometry (GC-MS/I MS) method after cleavage with beta-glucuronidase. The following pharmacokinetic variables were computed for the two formulations: maximum concentration (Cmax), time to maximum concentration (Tmax), half-life of elimination (t1/2), mean residence time (MRT), and areas under the plasma concentration-time curve (AUC(0-t) and AUC(0-infinity)). For calculation of the 90% confidence interval (CI), an analysis of variance (ANOVA) was carried out. The results indicated that treatment and subject had statistically significant effect on AUC(0-t), AUC(0-infinity), and Cmax, and the 90% CIs for AUC(0-t), AUC(0-infinity), and Cmax were outside the acceptable bioequivalence range. The relative bioavailability was 121.4 +/- 10.6% for AUC(0-infinity). Therefore, it can be concluded that the two formulations are not bioequivalent and the bioavailability of soft capsules is significantly higher than that of hard capsules.