Ehrlichia canis is a major tick-borne bacterial pathogen of dogs. Quantitative real-time PCR was evaluated for the detection of E. canis in naturally (NI) and experimentally infected (EI) dogs. DNA was extracted from blood, spleen and conjunctival swabs of experimentally infected dogs pre- and post-infection (PI), and during doxycycline therapy, and from blood and conjunctivas of naturally infected dogs. The primers and probe were designed to amplify a 93bp fragment of the single copy E. canis 16S rRNA gene with the TaqMan system. All EI dogs were positive for E. canis DNA by 7d PI and developed clinical ehrlichiosis by 9-12d PI. A rapid increase in ehrlichial DNA in EI dogs correlated with the appearance of severe clinical signs of disease. The mean spleen and blood DNA copies significantly increased by more than 10-folds from 7d PI to 10 and 12d PI (p<0.05). E. canis DNA was undetectable in the blood by day 9 post-treatment. Although the spleen was more frequently positive than blood (15/15 specimens vs. 13/15), no significant differences were found between the mean ehrlichial DNA copies in the spleen and blood on each day of examination. In 12 naturally infected dogs, the mean blood DNA copies was similar to the number found in EI 7d PI, but significantly lower than the means of 10 and 12d PI (p<0.0001). Although the conjunctivas of all EI dogs were positive by 12d PI, only 3/5 (60%) NI dogs were positive also by conjunctival PCR. In conclusion, the kinetics of E. canis during acute experimental infection with complete pathogen clearance following doxycyline treatment was demonstrated for the first time by real-time PCR. The value of real-time PCR was shown in NI dogs as well as in EI dogs with spleen and blood sampling more sensitive than non-invasive conjunctival PCR.