31P nuclear magnetic resonance of highly oriented DNA fibers has been observed for three different conformations, namely, the A, B, and C forms of DNA. At a parallel orientation of the fiber axis with respect to the magnetic field, DNA fibers in both the A and B forms exhibit a single, abnormally broad resonance; in contrast, fibers in the C form show almost the full span of the chemical shift anisotropy (170 ppm). The spectra of the fibers oriented perpendicular indicate that the DNA molecules undergo a considerable rotational motion about the helical axis, with a rate of greater than 2 x 10(3) s-1 for the B-form DNA. Theoretical considerations indicate that the 31P chemical shift data for the B-form DNA fibers are consistent with the atomic coordinates of the phosphodiester group proposed by Langridge et al. [Langridge, R., Wilson, H. R. Hooper, C. W., Wilkins, M. H. F., & Hamilton, L. D. (1960) J. Mol. Biol. 2, 19--37] but not with the corresponding coordinates proposed by Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Coomun. 47, 1504--1509], and also lead to the conclusion that the phosphodiester orientation must vary significantly along the DNA molecule. The latter result suggests that DNA has significant variations in its backbone conformation along the molecule.