We examined the metal ion cofactor preference for MST3 (mammalian Ste20-like kinase 3) of the Ste20 serine/threonine kinase family. Four metal ions (Mg+2, Mn+2, Zn2+, and Co2+) activate endogenous, exogenous, and baculovirus-expressed recombinant MST3 within the physiological concentration range. In contrast, Fe+2 and Ca+2 do not function as MST3 cofactors. Mn2+, Co 2+, and Mg2+-dependent autophosphorylation of MST3 is mainly on threonine residue while Zn2+-stimulated MST3 autophosphorylation is on both serine and threonine residues. The distinct autophosphorylation pattern on MST3 suggests that MST3 may exert various types of kinase reactions depending on the type of metal ion cofactor used. To our knowledge, this is the first report showing Zn2+ as the metal ion cofactor of a recombinant serine/threonine kinase.