We have isolated maize cDNAs encoding three manganese-containing superoxide dismutases (MnSODs) distinct from the one previously reported. Molecular analyses indicate that multiple MnSOD transcripts are encoded by different, though similar, genes in the maize genome. A single MnSOD gene has been reported in all other organisms examined to date. The deduced amino acid sequences show that these maize MnSOD proteins have a mitochondrial transit peptide and that the first 9 amino acids (matrix-targeting sequence) in the transit peptide are conserved. This suggests that all the maize MnSOD proteins are mitochondria-associated isozymes. RNA blot analysis demonstrated that each member of the maize MnSOD multigene family is both spatially and developmentally regulated. One gene, Sod3.3, was predominantly expressed in the embryo late in embryogenesis. Patterns of increased Mn-SOD transcript accumulation are shown to be associated with increased mitochondrial activity during plant growth and development. The influence of mitochondrial metabolism on the expression of the nuclear MnSOD genes is discussed.