Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca2+-binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the “blocked” and “free” heads of each myosin, inhibiting actin-binding or ATPase activity (Wendt et al., 2001; Woodhead et al., 2005). We have tested whether a similar interaction occurs in Ca2+-regulated filaments. Filaments were purified from scallop striated adductor muscle and imaged by cryo-electron microscopy. 3D reconstruction was carried out by single particle methods. Reconstructions showed a 7-fold symmetric, helical array of myosin head-pair motifs lying above the filament surface. Fitting of the motif with a myosin head atomic model revealed that the heads interact in a similar way to phosphorylation-regulated filaments. However, the 2-headed motif was more tilted and higher above the filament surface in the Ca2+-regulated filaments. Subfragment 2 of the myosin tail emerged from the motif near the blocked head and connected the motif to the filament backbone, which comprised a 7-fold array of comma-shaped subfilaments. This structure reveals new detail compared with a previous cryo-EM study (Vibert, 1992) and demonstrates that the interpretation of head organization in a negative stain reconstruction of scallop filaments (AL-Khayat et al., 2009) is incorrect. We conclude that the relaxed state of Ca2+-regulated filaments is achieved in a similar way to phosphorylation-regulated filaments, confirming that head-head interaction is a widely used motif (Woodhead et al., 2005). In the scallop filament, the pairs of myosin heads are much closer together azimuthally and the subfilaments have a different structure compared with phosphorylation-regulated filaments, implying that general models for thick filament structure (Squire, 1973) need modification. Supported by NIH grant AR34711.