A symbiotic association between leguminous roots and soil fixing nitrogen bacteria is required for legume nodule formation. The primary function of nodules is the fixation of nitrogen from the atmosphere into an accessible form for plants. In this study, nodules of plants of the soybean cultivar Prima 2000 were characterized and their number and weight were determined during nodule development. Their nitrogen-fixing activity during nodule development was determined by color evaluation. A pink nodule color showed active leghemoglobin required for nitrogen fixation and a green nodule color nonfunctional leghemoglobin. Strong appearance of nonfunctional leghemoglobin in the later stages of nodule development during senescence was accompanied by an increase in protease activity within crown nodules demonstrated by gelatine-containing SDS PAGE. Cysteine protease activity was identified as a major protease activity during nodule senescence when the cysteine protease inhibitor E-64 was used to block total protease activity. Products, which may indicate the expression of cysteine protease inhibitors during nodule development, were detected with the reverse zymogram technique and Western blotting. However, these bands have not been characterized so far in more detail. Putative transgenic plants were produced using the Agrobacterium transformation technique to allow determining the activity of native and mutated papaya cysteine protease inhibitor coding sequences. These sequences will ultimately be used for soybean transformation to reduce cysteine protease activity in nodules. However, the presence of coding sequence in the genome of these putative transgenic plants could not be confirmed by gene amplification and protease activity testing. Overall, this study has contributed to establish parameters to measure nodule growth and performance during development.