The development and use of single molecule methods in biophysics is rapidly expanding. One of the current emphases in the development of single molecule techniques is maximizing the information that can be extracted from the data. Several years ago, the group of Simon Weiss introduced the concept of alternating laser excitation [1], which we combined with subnanosecond pulsed lasers in the method of pulsed interleaved excitation (PIE) [2]. By using alternating excitation schemes, the labeling stoichiometry of can be determined, which is very advantageous for single-pair Forster Resonance Energy Transfer (spFRET) experiments.In the last years, the first single-molecule multicolor FRET experiments using alternating excitation have been performed [3-5]. The stoichiometry information can be used to distinguish incompletely labeled species from complexes that contain all labels. Here, we show how the different excitation wavelengths can be used to examine the presence of different components or the distance between subsets of the fluorophores. In addition, the stoichiometry information of the incompletely labeled species can be analyzed as well to independently measure FRET distances between various dye pairs and simplify the multicolor FRET analysis. We have applied multicolor FRET to investigate the presence of coordinated motions between the nucleotide and substrate binding domains of HSP70.[1] Kapanidis, A. N.; Lee, N. K.; Laurence, T. A.; Doose, S.; Margeat, E.; Weiss, S. PNAS 2004, 101, 8936.[2] Muller, B. K.; Zaychikov, E.; Brauchle, C.; Lamb, D. C. Biophysical journal 2005, 89, 3508.[3] Stein, I. H.; Steinhauer, C.; Tinnefeld, P. Journal of the American Chemical Society 2011, 133, 4193.[4] Lee, J.; Lee, S.; Ragunathan, K.; Joo, C.; Ha, T.; Hohng, S. Angew Chem Int Ed Engl 2010, 49, 9922.[5] Gambin, Y.; Deniz, A. A. Molecular BioSystems 2010, 6, 1540.