Nucleus HVC (formerly called high vocal center) of songbirds contains two types of projecting neurons connecting HVC respectively to the nucleus robustus archistriatalis, RA, or to area X. These two neuron classes exhibit multiple neurochemical differences and are differentially replaced by new neurons during adult life: high rates of neuronal replacement are observed in RA-projecting neurons only. The activity of these two types of neurons may also be modulated differentially by steroids. We analyzed by magnetic resonance imaging the effect of testosterone on the volume of RA and area X and on the dynamics of Mn(2+) accumulation in RA and area X of female starlings that had been injected with MnCl(2) through a permanent cannula implanted in HVC. Repeated visualization 6 weeks apart (before and after testosterone treatment) identified a volume increase of both nuclei in testosterone-treated birds associated with a concomitant decrease in controls. Following testosterone treatment, the total amount of Mn(2+) transported to RA and area X increased but the dynamics of accumulation, reflecting in part the activity of HVC neurons, was specifically altered in area X but not in RA. These data indicate that testosterone differentially affects the RA- and area X-projecting neurons in HVC. Manganese-enhanced magnetic resonance imaging (ME-MRI) thus provides repeated measures of connected brain areas and demonstrates testosterone-dependent regionally specific changes in brain activity and functional connectivity. The slow time scales investigated by this technique (compared to functional MRI) appear ideally suited for characterizing slow processes such as those involved in brain plasticity and learning.