Sequence-specific assignments of 1H NMR resonances were obtained for the backbone protons in acyl carrier protein (ACP) from Escherichia coli, a protein of 77 residues. The observations, in the NOESY spectra, of 1H-1H sequential and medium-range connectivities indicate the presence of three or four alpha-helical segments joined by short sequences of mixed conformations. The observations are used to refine a secondary structure model previously proposed on the basis of a Chou-Fasman algorithm [Rock, C. O., & Cronan, J. E., Jr. (1979) J. Biol. Chem. 254, 9778-9785].