High-spin Fe1+ sites are potentially important in iron-sulfur proteins but are rare in synthetic compounds and unknown in metalloproteins. Here, we demonstrate a spectroscopically characterized example of high-spin non-heme Fe1+ in a protein environment. Cryoreduction of Fe2+-substituted azurin at 77 K wit Co-60 gamma radiation generates a new species with a S = 3/2 (high-spin) Fe1+ center having D > 0 and E/D similar to 0.25. This transient species is stable in a glycerol-water glass only up to similar to 170 K. A combination of electron paramagnetic resonance and Mossbauer, spectroscopies provides a powerful means of identifying a transient high-spin Fe1+ site in a protein scaffold.