The high-molecular-weight RNA of Moloney murine leukemia virus (MuLV) was analyzed by sedimentation equilibrium ultracentrifugation. Molecular weights of 7.2 x 10(6) and 3.4 x 10(6) were found for the native and subunit forms, respectively, indicating that the native structure is a dimer. S20,w and frictional coefficients were determined for MuLV RNA by analytical velocity centrifugation as a function of ionic strength. The apparent S20,w of native MuLV RNA was 47.3, 57.4, and 66.5 in 0.01, 0.1, and 0.20 M Na+, respectively; the corresponding frictional coefficients were 5.44, 4.48, and 3.87. Native RNA was estimated by circular dichroism to be 85% helical, whereas denatured RNA was 54% helical. Thermal denaturation profiles were obtained from uv absorbance scans. Melting temperatures of 57 and 68 C were obtained for high-molecular-weight RNA in 0.01 M Na+ and 0.122 M Na+, 1mM Mg2+, respectively. van't Hoff plots of the thermal denaturation data gave enthalpies for the helix-coil transition of 21,600 cal (ca. 90,500 J) per mol of cooperatively melting unit in high salt and 19,600 cal (ca. 82,100 J) per mol in low salt, consistent with both base stacking and pairing. The melting of Mu LV RNA occurred over a broad temprange and van't Hoff plots were linear over most of the melting range, indicating a noncooperative process of helix stabilization.