Two different aconitases are known to be expressed after the germination of oil-seed plants. One is a mitochondrial aconitase that is involved in the tricarboxylic acid cycle. The other participates in the glyoxylate cycle, playing a role in gluconeogenesis from stored oil. We isolated and characterized a cDNA for an aconitase from etiolated pumpkin cotyledons. The cDNA was 3,145 bp long and capable of encoding a protein of 98 kDa. N-terminal and C-terminal amino acid sequences deduced from the cDNA did not contain mitochondrial or glyoxysomal targeting signals. A search of protein databases suggested that the cDNA encoded a cytosolic aconitase. Immunoblotting analysis with a specific antibody against the aconitase expressed in Escherichia coli revealed that developmental changes in the amount of the aconitase were correlated with changes in levels of other enzymes of the glyoxylate cycle during growth of seedlings. Further analysis by subcellular fractionation and immunofluorescence microscopy revealed that aconitase was present only the cytosol and mitochondria. No glyoxysomal aconitase was found in etiolated cotyledons even though all the other enzymes of the glyoxylate cycle are known to be localized in glyoxysomes. Taken together, the data suggest that the cytosolic aconitase participates in the glyoxylate cycle with four glyoxysomal enzymes.