In a prospective study of posttransfusion hepatitis, 14 patients who were diagnosed with posttransfusion hepatitis C were enrolled randomly for the study of hepatitis C virus (HCV) RNA in saliva. Saliva and serum samples were collected on the same day. Spouses of 11 married patients were also tested for anti-C100 and HCV RNA. Paired serum and saliva samples were tested for HCV RNA by a nested polymerase chain reaction (PCR). Two primer pairs specific for the non-coding region of HCV were used for the PCR and a oligonucleotide sequence between the primers was used as the probe for Southern hybridization. Six patients were positive for HCV RNA by first round PCR amplification and an additional four patients were detected after second round PCR. All patients were negative for HCV RNA in saliva after first round PCR, while seven were positive after second round PCR amplification. All seven patients were positive for HCV RNA in paired serum samples. HCV RNA was detectable in saliva from 1 week to 38 months after the onset of hepatitis. All spouses were negative for anti-C100 and HCV RNA. We conclude that HCV RNA is present in the saliva of approximately half of patients with acute and chronic hepatitis C, and the presence of HCV RNA correlates with HCV viremia. The efficiency of HCV transmission is low among spouses.