Abstract. To begin to understand the nature of myosin subunit assembly, we determined the region of a vertebrate sarcomeric myosin heavy chain required for binding of light chain 1. We coexpressed in Escherichia coli segments of the rat alpha cardiac myosin heavy chain which spanned the carboxyl terminus of subfragment 1 and the amino terminus of subfragment 2 with a full-length rat cardiac myosin light chain 1. A 16 amino acid region of the myosin heavy chain (residues 792-808) was shown to be required for myosin light chain 1 binding in an immunoprecipitation assay.M YosiN is an asymmetric hexamer comprised of two heavy chains (210 kD) and two pairs of light chains (18-25 kD). In smooth muscle, invertebrate muscle and vertebrate nonmuscle myosin, myosin light chain 2 (MLC2) ~ regulates the actin-activated ATPase of myosin in response to the calcium concentration of the cell (for reviews see Kamm and Stull, 1985;Citi and Kendrick-Jones, 1988). The role of MLC1 in myosin function is less clear (see Wagner and Giniger, 1981;Sivaramakrishnan and Burke, 1982). However, isoforms of MLC1 have been shown to be associated with different shortening velocities of skeletal muscle (Sweeney et al., 1988).The interactions of the subunits of myosin are not precisely defined, but both biochemical and electron microscopic studies suggest that MLCs bind near the junction of the subfragments 1 and 2 ($1 and $2). Scallop myosin reacted with anti-light chain antibodies demonstrates MLC2 binding near the head-rod junction (Flicker et al., 1983). Similar positions were noted for vertebrate MLC2, with MLC1 binding distally to MLC2, in the S1 head (Tokunaga et al., 1987;Katoh and Lowey, 1989). Proteolytic peptides of myosin heavy chain (MHC) from the carboxyl terminus of the S1 head bind MLC (Szentkiralyi, 1984;Mitchell et al., 1986). Burke et al. (1983) also implicated the carboxyl terminus of the S1 head by abolishing MLC1 binding with proteolytic digestion of a 3-kD segment from the S1-$2 junction. Sellers and Harvey (1984) used gel overlay to assign the light chain binding sites to 26 kD at the carboxyl terminus of smooth muscle myosin $1. However, Okamoto et al. (1986) have shown that MLC1 binds near the site of ATP binding on smooth muscle myosin. In the scallop it has been shown that exchange of MLC1 with exogenously added MLC1 can occur only in the absence of MLC2 (Ashiba and Szent-Gyorgyi, 1985). This finding, coupled with the observation that the 1. Abbreviations used in this Paper: MHC, myosin heavy chain; MLC, myosin light chain; S1 and $2, subfragment 1 and 2, respectively. sulfhydryl groups of scallop MLC1 are only available for labeling in the absence of MLC2, suggests that MLC1 lies between MLC2 and MHC (Walliman and Szent-Gyorgyi, 1981;Hardwicke et al., 1982).The use of recombinant DNA methods to produce myosin segments that have precisely defined sequences would be of great use in mapping the interactions of myosin subunits. A portion of the Caenorhabditis elegans unc54 MHC gene corresponding to S1 was expressed in Es...