The brainstem nucleus incertus (NI) plays an important role in processing aversive stimuli and modulating stress response. In the midbrain, apart from the ventral tegmental area (VTA), the NI also innervates the rostromedial tegmental nucleus (RMTg) – the main inhibitory input to the dopaminergic (DA) system in mammals. In order to better understand the circuit’s anatomy, retrogradely transported viral vectors, carrying genes for fluorescent proteins, were unilaterally injected into the RMTg of Sprague-Dawley rats. The results show that the RMTg, unlike the VTA, receives bilateral innervation from the NI. Notably, only a minority of these neurons contain relaxin-3, a neuropeptide synthesized by the best-known subpopulation of NI neurons. Subsequently, electrophysiological recordings in urethane-anesthetised rats were performed, preceded by injections of two viral vectors: one retrograde vector carrying Cre recombinase gene and another carrying Cre-dependent genes for an excitatory opsin. These injections targeted the VTA or RMTg and NI, respectively. Results confirmed that the NI controls midbrain DA neurons via an inhibitory innervation of the VTA. Additionally, the functional impact of robust NI-RMTg innervation was verified. Lastly, prior to behavioural experiments, retrograde viral vectors carrying YFP were injected into the animals’ RMTg bilaterally. Following a stress-induction procedure in operant conditioning chamber, a series of immunostaining was performed. The resulting data provide insight into the presumed overlap between c-fos-reactive NI subpopulation and the neurons innervating the RMTg. In summary, our study highlights that NI-originating innervation of the midbrain structures forms a complex system involved in stress and reward processing.Funding: National_Science_Centre: OPUS_17_2019/33/B/NZ4/03127,Preludium_21_2022/45/N/NZ4/03171