Donor-Donor Energy Migration (DDEM) and fluorescence anisotropy experiments can be utilized as a versatile tool for examining protein structure and function. For this, pairs of identical fluorescent probes (D) are attached to unique residues created by means of site specific mutagenesis. Present work illustrates the applicability of the method on the latent form of Plasminogen Activator Inhibitor-1 (PAI-1). Different DD-pairs of mutated PAI-1 were prepared and studied, namely; V106C-H185C, H185C-M266C and M266C-V106C. The Cys residues were labelled with a sulfhydryl specific derivative of BODIPYR [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-yl)methyl iodo-acetamide]. To determine the rate of DDEM within such a pair, intramolecular order and dynamics must be considered (Biophys. J., 74, 11-21, 1997). For analysis of data, additonal information was obtained from experiments with the corresponding D-labelled single Cys mutants, that is, V106C, H185C and M266C. The stability of values determined was tested by generating and re-analyzing synthetic data. The intramolecular distances obtained agree, reasonably well, with those determined from the X-ray structure of latent PAI-1.