Metallothionein-3 (MT-3), or neuronal growth inhibitory factor, which exhibits growth inhibitory activity, is a brain-specific metallothionein. In this study, the effect of nitric oxide (NO) on metal release (using Cd2+ as a probe) from MT-3 was examined by 113Cd and 2D [1H-15N] heteronuclear single-quantum coherence NMR spectroscopy. The exposure of human MT-3 to NO leads to a nonselective release of the three metals from the beta-domain. In contrast to metallothionein-1 and metallothionein-2, two of the bound metals in the alpha-domain were also partially released, with the domain structure remaining almost unchanged. Further addition of NO resulted in the complete release of metals and concomitant unfolding of the protein. The preference of release of the two metals in the alpha-domain was attributed to the presence of two slightly different coordination environments for the four cadmium/zinc atoms.