The first X-ray crystallographic study for the alkaline structure transition of cytochrome c' was performed. Precise X-ray crystal structures of acidic (pH 6.0) and alkaline (pH 10.4) iron(111) cytochrome c' from Alcaligenes xylosoxidans NCIMB 11015 (AxCyt c') were determined at 1.1 and 1.48 angstrom resolution, respectively. The structure of AxCyt c' at pH 10.4 provides direct evidence for the alkaline high-spin transition caused by bond length elongation between the heme iron and the axial His 120 imidazole nitrogen atom.