We have developed a protocol for the sequential release of the intermembrane space (IMS) content of Saccharomyces cerevisiae mitochondria. Two distinct fractions were obtained: a soluble IMS with cytochrome b2 as key marker and a salt-extractable IMS with cytochrome c as key marker. The identity of several proteins was determined by amino-terminal amino acid sequencing. The IMS fractions were devoid of contaminations from cytosol and mitochondrial outer and inner membranes. By subtraction analysis, the protein profiles of soluble and salt-extractable IMS fractions were depleted of contaminating bands derived from matrix proteins. The fractionation method will provide the basis for the further analysis of IMS proteins and characterization of their functions in bioenergetics, mitochondrial biogenesis, and regulatory processes.