The reactive thiol of the myosin head, SH-1, can be selectively labelled in glycerinated rabbit muscle fibres. This residue has been used as an attachment site for either fluorescent or spectroscopic probes which report on head movements and orientations in various functional states of muscle. We have specifically modified SH-1 in vitro, using purified rabbit myosin and conditions similar to those employed in the labelling of muscle fibres (low ionic strength [40 mM NaCl] at 4°C), with stoichiometric amounts of either [14C]-iodoacetamide, 5-(2((iodoacetyl)amino)ethyl) aminonaphthalene-1-sulphonic acid (IAEDANS), or 4-(2-iodoacetamido-2,2,6,6-tetramethyl piperidinooxyl (IASL). The specificity of modification was determined by measuring the well-defined alterations in the high salt ATPase activities of myosin and by localizing both IAAm and IAEDANS to the 20-kDa C-terminal subfragment 1 (S1) which contains SH-1. The low ionic strength actin-activated Mg2+-ATPase of SH-1-modified rabbit myosin was measured in the presence of the thin filament regulatory, complex, troponin-tropomyosin. A significant increase in this activity in the absence of calcium, concomitant with a decrease in activity in the presence of calcium, was observed as the extent of SH-1 modification was incrementally increased from zero to one mole of label bound per mole of SH-1. The elevated myosin Mg2+-ATPase, which results from SH-1 modification, does not account for the increased actin-activated Mg2+-ATPase in resting conditions (i.e. in the absence of calcium). The in vitro actin-activated Mg2+-ATPase activities become equal in both active and resting conditions when one mole of SH-1 is modified per mole of myosin head. These results demonstrate that SH-1 is located in a region of the myosin head which plays a part in the calcium-sensitive regulation of the actin-activated Mg2+-ATPase by troponin-tropomyosin. These studies also indicate that SH-1-labelled preparations may not be suitable for the analysis of myosin head motion and/or orientation in the resting state.