DNA.RNA hybrid duplexes are found in many important biological processes and are involved in developing modes of disease treatment, such as antisense therapy, yet little is known about the sequence dependence of their structure and stability. The structure and thermodynamic stability of DNA.RNA hybrid model systems corresponding in composition and length and containing (1) all purine or all pyrimidine bases on each strand or (2) mixed purine and pyrimidine bases on each strand have been evaluated relative to pure RNA and DNA duplexes by thermal melting, CD, and electrophoresis analyses. The spread in free energies of denaturation of the homopurine.homopyrimidine systems covers over 14 kcal/mol of single strands, while the mixed sequence free energies vary by less than 4 kcal/mol. The RNA-homopurine.DNA-homopyrimidine hybrid resembles a corresponding pure RNA duplex in both structure and stability, whereas the DNA-homopurine.RNA-homopyrimidine hybrid resembles a corresponding pure DNA duplex. The mixed sequence hybrids show intermediate structure between the corresponding pure RNA and pure DNA duplexes and a stability closer to that of the pure DNA duplex. From these results and the evaluation of published hybrid data [Hall, K. B., & McLaughlin, L. W. (1991) Biochemistry 30, 10606-10613; Roberts, W. R., & Crothers, D. M. (1992) Science 258, 1463-1466], it can be predicted that a hybrid duplex containing more RNA purine bases will have a CD spectrum, and probably conformation, resembling that of A-form duplexes and will be more stable than a corresponding hybrid duplex with fewer RNA purine bases.