31P NMR of salmon sperm DNA, poly(rA).cntdot.poly(rU), and poly(rA).cntdot.poly(dT) fibers were measured as a function of relative humidity. The results indicated that the spectra were strongly perturbed by the molecular motions occurring in the hydrated fibers. The humidity dependence of the spectra at a number of orientations of the fibers relative to the magnetic field was reasonably explained by taking into account at least 3 motional modes, i.e., conformational fluctuations, restricted rotation about a tilted axis, and rotational diffusion about the helical axis. The rotational diffusion about the helical axis was found to perturb the spectral line shapes most strongly, and its constants were 1.5 .times. 104 and 5.0 .times. 104 s-1 for DNA fibers at 92% and 98% relative humidities, respectively. A DNA-RNA hybrid, poly(rA).cntdot.poly(dT), has been shown to adopt different conformations on 2 strands at a high relative humidity [S. B. Zimmerman and B. H. Pheiffer (1981)], which was unquestionably confirmed in the present study: i.e., the 31P NMR spectra from the hydrated form of this polymer were clearly explained by assuming that one strand had an A-like conformation and the other a B-like conformation.