We have used a new hybridization assay for the detection of the genome of hepatitis delta virus (HDV) in serum using a strand-specific RNA probe obtained by transcription of a recombinant riboprobe. This assay was tested on a panel of 30 sera from HBsAg carriers with hepatitis delta antigen (HDAg) in the liver. The riboprobe assay detected HDV RNA in the serum of 83% of the patients, while 63% were positive using the DNA hybridization assay. HDAg was detected in 73% of the same sera by immunoblotting. The riboprobe assay was also compared to other assays on serial samples from an experimentally infected chimpanzee. These results demonstrate that the Northern blot assay using the RNA probe is more sensitive than the homologous DNA probe for the detection of HDV in serum and is also more sensitive than the immunoblot assay for HDAg. The riboprobe assay is the most sensitive of currently available methods to measure HD viremia.