Microelectrode recordings of single unit neuronal activity were used during stereotactic surgery to define the subthalamic nucleus for chronic deep brain stimulation in the treatment of Parkinson's disease. By using five parallel trajectories, often two to three microelectrodes allow us to recognize subthalamic nucleus (STN) neuronal activity. STN neurons were easily distinguished from cells of the overlying zona incerta and the underlying substantia nigra. During a typical exploratory track, we can observe a very low background noise in the zona incerta and almost complete absence of single cell recording. Penetration of the electrode tip into the STN is characterized by a sudden increase in background activity and single cell activity of spontaneously active neurons. The exit of electrode tip out of the STN corresponds to a decrease in background noise and a loss of single cell activity. Spontaneous neuronal activity increases again when the electrode tips enters the substantia nigra pars reticulata (SNr); however, the activity is less rich than in the STN, indicating a more cell‐sparse nucleus. STN neurons are characterized by a mean firing rate of 42.30 ± 22.00 spikes/sec (mean ± SD). The STN cells exhibited irregular or bursty discharge pattern. The pattern of single cell activity in the SNr is a more regular tonic activity that can easily be distinguished from the bursting pattern in the STN. The most useful criteria to select a trajectory are (1) the length of an individual trajectory displaying typical STN activity, (2) the bursting pattern of activity, and (3) motor responses typical of the sensorimotor part of the nucleus. In conclusion, microelectrode recording of the subthalamic area improves the accuracy of targeting the STN. © 2002 Movement Disorder Society