Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics. If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the "second" genetic code problem would be solved. Regrettably, no such technique currently exists. As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Förster resonance energy transfer [Lillo, M. P., et al. (1997) Biochemistry 36, 11261-11272]. In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK. These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-[[[(2-iodoacetyl)amino]ethyl]amino]naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor). The unfolding of PGK is found to be a sequential multistep process (native --> I1 --> I2 --> unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded). Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1 and I2 states. With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A. As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study [Beechem, J. M., et al. (1995) Biochemistry 34, 13943-13948]. Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5-10 A.