In the present study hydrostatic pressure was applied upon both skeletal myosin rod molecules and rod minifilaments to learn more of the intra- and intermolecular interaction behavior of myosin. Applied pressure disassembled the rod minifilaments into individual rod molecules and dissociated each myosin rod molecule into two chains of alpha-helix. The dissociation and disassembly profiles of these systems were obtained by measuring their fluorescent anisotropy under pressure. The mid-disassembly pressure of rod minifilaments at 0.4 mg/mL concentration was 430-490 bar. However, dissociation of two helical strands of rod molecules occurred at a much higher pressure, with a mid-disassembly pressure of 1300 bar at this concentration. These results indicate that the intramolecular interactions occurring between two alpha-helical chains of a rod molecule are much more stable under pressure than the intermolecular interactions that occur among rod molecules in a minifilament. The regions in the rod molecules involved in filament assembly were investigated through usage of both the intrinsic fluorescence of tryptophan residues and the extrinsic fluorescence of 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) labeled cysteine residues. The blue spectral shifts upon minifilament formation suggest the participation of both light meromyosin (LMM) and subfragment-2 (S-2) regions of myosin rods in the filament formation. Profiles of thermal unfolding of myosin rod molecules and rod minifilaments were obtained by circular dichroism measurement. The multiple transitions exhibited upon unfolding profiles indicated the presence of more than one structural domain, each correlating with a cooperative transition. The domain transitional temperatures were found to be 1-4 degrees C higher for rods in minifilaments than those for rod molecules in a solution of similar ionic composition, indicating that all structural domains are involved in filament assembly. Furthermore, the domain transitional temperatures for rod molecules in a buffer containing 0.6 M NaCl were 6-8 degrees C higher than those for rod molecules in 5 mM sodium pyrophosphate buffer, suggesting that each structural domain of a rod molecule becomes stabilized at 0.6 M NaCl solution.