Single-pulse transcranial magnetic stimulations (TMSs) of the motor cortex (M1) were performed in order to decipher the neural mechanisms of time preparation. We varied the degree to which it was possible to prepare for the response signal in a choice reaction time (RT) task by employing either a short (500 ms) or a long (2500 ms) foreperiod in separate blocks of trials. Transcranial magnetic stimulations were delivered during these foreperiods in order to study modulations in both the size of the motor evoked potential (MEP) and the duration of the silent period (SP) in tonically activated response agonists. Motor evoked potential area and silent period duration were assumed to reflect, respectively, the excitability of the cortico-spinal pathway and the recruitment of inhibitory cortical interneurons. Shorter reaction times were observed with the shorter foreperiod, indicating that a better level of preparation was attained for the short foreperiod. Silent period duration decreased as time elapsed during the foreperiod and this decrement was more pronounced for the short foreperiod. This result suggests that time preparation is accompanied by a removal of intracortical inhibition, resulting in an activation. Motor evoked potential area decreased over the course of the short foreperiod, but not over the long foreperiod, revealing that time preparation involves the inhibition of the cortico-spinal pathway. We propose that cortico-spinal inhibition secures the development of cortical activation, preventing erroneous premature responding.