The DNA hairpins d[CGATCG-Tn-CGATCG] (n = 3, 4) have been studied by NMR in order to gain information on hairpin conformation and flexibility. Resonance assignments were made using a combination of DQF-COSY, DQF-COSY[31P], NOESY, and 1H-31P-COSY. These data also provide approximate coupling constant information which points out exceptionally flexible regions of the phosphate backbone. The data for both hairpins reveal substantial flexibility within the loop segments. For n = 4, NOESY data alone are insufficient to distinguish between two loop-folding motifs, although coupling constant data favor a conformation in which Tb is folded toward the minor groove and is highly exposed to solvent. This is in agreement with chemical shift data and susceptibility to modification by KMnO4. The phosphate backbone between Tc and Td is exceptionally flexible, undergoing a facile exchange between (beta t,gamma+) and (beta+,gamma t) conformers. A similar flexible phosphate is observed between Tc and C7 when n = 3. Differences in stem conformation and dynamics in both hairpins are restricted to the two base pairs adjacent to the stem-loop junction. The C7pG8 stem phosphate appears to flip easily between (zeta-,alpha-) and (zeta-,alpha t) conformers when n = 4 but not when n = 3. Hairpin loop size thus affects the conformational flexibility of the adjacent stem segment.