Genetic variability is an important consideration in the study of the effect of drugs on humans and animals. Specifically, the effect of drugs on behavior varies from one rat strain to another. Methylphenidate (MPI), commonly known as Ritalin, is a psychostimulant with a pharmacological profile similar to amphetamine and cocaine, and is widely used in the treatment of attention deficit/hyperactivity disorder (ADHD). The purpose of this study was to examine the acute dose-response characteristics of MPD on three different male rat strains: spontaneously hypertensive/hyperactive rats (SHR), Wistar-Kyoto (WKY) rats, and Sprague-Dawley (SD) rats. Results showed clear differences in the pattern of behavioral response to the various MPD doses among the three rat strains. The 0.6 mg/kg MPD dose had no effect on any of the strains. At the dose of 2.5 mg/kg MPD, WKY rats reached peak locomotor activity within the initial 10 min, whereas SHR and SD rats reached peak activity in approximately 30 min. Furthermore, the effects on locomotor activity of SHR were longer in duration and higher in locomotor activity, whereas the effects on SD rats were shorter in duration and amplitude. Following the 10.0 mg/kg MPD, WKY, and SD mats reached their peak locomotor activity within 80 min post-injection, whereas SHR reached their peak locomotor activity within the initial 10 min post-injection. In addition, the SHR strain exhibited increased behavioral sensitivity to MPD as expressed by the latency, duration, and amplitude of their response. In conclusion, the dose-response experiment on the acute effects of MPD demonstrated that there are genetic differences in the responses to MPD.