The identity and location of vocalization pattern generating (VPG) circuits in mammals is debated. Based on physiological experiments investigators suggested anterior brainstem circuits in the reticular formation, and anatomical evidence suggested the nucleus retroambiguus (NRA) in the posterior brainstem, or combinations of these sites as the putative mammalian VPG. Additionally, vocalization loudness is a critical factor in acoustic communication. However, many of the underlying neuronal mechanisms are still unknown. Here we evoked calls by stimulation of the periaqueductal gray (PAG) in anesthetized male rats, performed a large-scale mapping of vocalization-related activity using the activity marker c-fos, and high-density recordings of brainstem circuits using Neuropixels probes. Both c-fos expression and recording of vocalization-related activity point a participation of the NRA in vocalization. More importantly, among our recorded structures, we found that the NRA is the only brainstem area showing a...