We have previously shown that the orientation of (iodoacetamido)tetramethylrhodamine labels on SHj thiol of S-l moieties changes when MgADP is added to the fibers in rigor [Borejdo, J., Assulin, O., Ando, T., & Putnam, S. (1982) J, Mol Biol. 158, 391-414. Burghardt, T. P., Ando, T., & Borejdo, J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515-7519]. Here we report the results of experiments in which the SH2 of S-l was labeled with maleimidorhodamine. The specificity of modification of thiols was checked by measuring the stoichiometry of attached dye, by determining the extent of the decrease in EDTA(K+)- and Ca2+-ATPase activities, and by the localization of the dyes on peptides containing SH1 and/or SH2. Labeled S-1 was diffused into single glycerinated fibers of rabbit psoas muscle, and the orientation of chromophores was measured by fluorescence detected dichroism. The dye attached to SH1 was oriented at 650 with respect to the fiber axis in rigor and at 51 ° in the presence of MgADP, regardless of whether SH2 was modified or not. The dye on SH2 was oriented near 42° both in the presence and in the absence of ADP, regardless of whether SH1 was modified or not. Our results show that rhodamine oriented differently when attached to SH2 compared with when attached to SH1 and that in the former placement it was not sensitive to MgADP. We think this indicates that the SH2-containing region has a mobility different from that of the SH1-containing region, i.e., that this is evidence for internal flexibility of S-1.