A putative secondary structure of the mRNA for the human hepatitis B virus (HBV) X gene is proposed based on not only chemical and enzymatic determination of its single- and double-stranded regions but also selection by the computer program MFOLD for energy minimum conformation under the constraints that the experimentally determined nucleotides were forced or prohibited to base pair. An RNA of 536 nucleotides including the 461-nucleotide HBV X mRNA sequence was synthesized in vitro by the phage T7 RNA polymerase transcription. The thermally renatured transcripts were subjected to chemical modifications with dimethylsulfate and kethoxal and enzymatic hydrolysis with single strand-specific RNase T1 and double strand-specific RNase V1, separately. The sites of modification and cleavage were detected by reverse transcriptase extension of 4 different primers. Many nucleotides could be assigned with high confidence, twenty in double-stranded and thirty-seven in single-stranded regions. These nucleotides were forced and prohibited, respectively, to base pair in running the recursive RNA folding program MFOLD. The results suggest that 6 different regions (5 within X mRNA) of 14 similar to 23 nucleotides are single-stranded This putative structure provides a good working model and suggests potential target sites for antisense and ribozyme inhibitors and hybridization probes for the HBV X mRNA.