HVC (nidopallial area, formerly known as hyperstriatum ventrale pars caudalis), a key centre for song control in oscines, responds in a selective manner to conspecific songs as indicated by electrophysiology. However, immediate–early gene induction cannot be detected in this nucleus following song stimulation. HVC contains neurons projecting either towards the nucleus robustus archistriatalis (RA; motor pathway) or area X (anterior forebrain pathway). Both RA- and area X-projecting cells show auditory responses. The present study analysed these responses separately in the two types of HVC projection neurons of canaries by a new in vivo approach using manganese as a calcium analogue which can be transported anterogradely and used as a paramagnetic contrast agent for magnetic resonance imaging (MRI). Manganese was stereotaxically injected into HVC and taken up by HVC neurons. The anterograde axonal transport of manganese from HVC to RA and area X was then followed by MRI during ≈ 8 h and changes in signal intensity in these targets were fitted to sigmoid functions. Data comparing birds exposed or not to conspecific songs revealed that song stimulation specifically affected the activity of the two types of HVC projection neurons (increase in the sigmoid slope in RA and in its maximum signal intensity in area X). Dynamic manganese-enhanced MRI thus allows assessment of the functional state of specific neuronal populations in the song system of living canaries in a manner reminiscent of functional MRI (but with higher resolution) or of 2-deoxyglucose autoradiography (but in living subjects).