The conventional assay procedure for cathepsin D (E.C.188.8.131.52) activity in tissue homogenates and subcellular fractions requires incubation with hemoglobin as substrate. Cathepsin D (CD) activity is calculated by determining the increase in absorbance at 280 nm after precipitation of all proteins with trichloroacetic acid. This increase in absorbance (presumably due to the release of tyrosine residues from hemoglobin) is converted to arbitrary CD activity units. Homogenization and fractionation of cardiac tissue frequently requires that ethylenediamine tetraacetic acid (EDTA) be included in the homogenization medium. We have observed that subcellular fractions of cardiac tissue prepared in the presence of EDTA demonstrate residual CD activity despite either quantitative removal of all CD protein by immunoprecipitation or complete inhibition of CD by pepstatin. The present study demonstrates that this ‘apparent’ CD activity (residual increase in absorbance at 280 nm) is due to the formation of an Fe-EDTA complex which absorbs at 280 nm. Data are presented which demonstrates that the EDTA of the medium complexes with non-heme iron which contaminates commercially available hemoglobin. A method for preparing hemoglobin free of contaminant non-heme iron is described for use in studies of CD metabolism when EDTA is present in the homogenization buffer.