Background: Currently, the underlying neurobiological mechanism as to how repetitive transcranial magnetic stimulation (rTMS) can alter depressive states remains unclear. Animal data suggest that its influence could occur at the neurotransmitter level, such as modulation of the serotonin system. Methods: Twenty-one antidepressant-free medication-resistant unipolar depressed patients, and 21 age- and gender-matched healthy subjects were studied. We examined the neurobiologic impact of 10 high-frequent (HF)-rTMS sessions applied to the left dorsolateral prefrontal cortex (DLPFC) on postsynaptic 5-HT2A receptor binding indices (BI) measured with 123I-5-I-R91150 single photon emission computed tomography (SPECT) only in patients. Results: Compared with the control group, patients displayed significantly less bilateral dorsolateral prefrontal cortical and significantly higher left hippocampal baseline 5-HT2A receptor BI. Successful HF-rTMS treatment correlated positively with 5-HT2A receptor BI in the DLPFC bilaterally and correlated negatively with right hippocampal 5-HT2A receptor uptake values. Conclusions: Our results indicate that HF-rTMS treatment affect the serotonergic system. Our data also suggest that this kind of treatment affects 5-HT2A receptor BI in the DLPFC and in the hippocampus in different ways.