We have utilized fluorescence resonance energy transfer (FRET) to investigate the spatial proximities of segments in the Msx-1 homeodomain (Msx). This strategy makes use of a single, invariant tryptophan (Trp-48) in helix III as the donor for FRET. The acceptor molecule, 5-[[[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (AEDANS), was incorporated into Msx at positions 6, 10, or 27 which are within the N-terminal arm, and helices I and II since these segments have been implicated in interactions with helix III. Specific incorporation of AEDANS was achieved by using a two-step strategy consisting of site-directed mutagenesis for introducing unique cysteine residues at the selected positions followed by covalent modification of these cysteine residues with AEDANS. Using this approach, we demonstrated energy transfer between Trp-48 and the AEDANS-labeled cysteines at positions 6, 10, and 27 and estimated the distances between the Trp-48 and AEDANS pairs to be 19, 23, and 16 A, respectively. We further demonstrated that FRET provides a strategy for detecting subtle alterations in protein conformation that result from replacement of specific residues in helix III and the N-terminal arm. Together, these findings show that FRET provides a useful approach for estimating intramolecular distances and for examining the conformation of Msx. Moreover, given the fact that Trp-48 is invariant among all homeodomain sequences, we propose that FRET will provide a general approach for facilitating comparative analyses of homeodomain conformations.