RNA–DNA hybrid duplexes are involved in transcription, replication and reverse transcription of nucleic acids. Information on such duplexes may shed some light on the mechanism of these processes. For this purpose, the influence of base composition on the structure of a polypyrimidine–polypurine RNA–DNA duplex r(cucuccuucucuu). d(GAGAGGAAGAGAA) has been studied using 1H, 31P and 13C NMR experiments, molecular modeling (JUMNA program) and NOE back-calculation methods. The resulting structure of the 13-mer hybrid duplex shows that the RNA strand is in the expected A-type conformation while the DNA strand is in a very flexible conformation. In the DNA strand, the desoxyribose sugars retain the C2%-endo B-type conformation. The duplex helical parameters (such as inclination, twist and displacement of the bases) are close to the A-type conformation. No bending was observed for the global axis curvature. The major groove width is close to the B-form value and the minor groove width is intermediate between standard values for A and B-forms. These results are in favour of the independence of minor groove size (where RNase H interacts) and the base composition of the hybrid duplexes. © 2001 Elsevier Science B.V. All rights reserved.