BACKGROUND: Human herpesvirus type 8 (HHV-8) is hyperendemic in Amerindian populations, but its modes of transmission are unknown. METHODS: Antibodies against either HHV-8 lytic antigen or HHV-8 latency-associated nuclear antigen (LANA) were detected, by immunofluorescence assays, in 339 Amerindians and 181 non-Amerindians from the Brazilian Amazon. Serological markers of oro-fecal (hepatitis A), parenteral (hepatitis B and C), and sexual (herpes simplex virus type 2 and syphilis) transmission were measured by specific ELISAs. Salivary HHV-8 DNA was detected by use of a nested polymerase chain reaction assay and was sequenced. RESULTS: Antibodies against either lytic antigen or LANA were detected in 79.1% of Amerindians and in 6.1% of non-Amerindians (adjusted seroprevalence ratio [SR], 12.63 [95% confidence interval {CI}, 7.1-22.4]; P<.0001). HHV-8 seroprevalence increased with age among Amerindians (P(Trend) < .001) and already had high prevalence in childhood but was not sex specific in either population. The 2 populations did not differ in seroprevalence of oro-fecal or parenteral markers, but seroprevalence of markers of sexual transmission was lower among Amerindians. HHV-8 DNA in saliva was detected in 47 (23.7%) of 198 HHV-8 seropositive Amerindians. Detection of HHV-8 DNA decreased with age (P(Trend) < .04) and was more common in men (SR, 2.14 [95% CI, 1.3-3.5]; P=.003). A total of 36 (76.6%) of the 47 saliva HHV-8 DNA samples were sequenced, and all clustered as subtype E. CONCLUSION: The data support the hypothesis of early acquisition and horizontal transmission, via saliva, of HHV-8 subtype E in Amerindian populations.