Twenty-four adult Beechey ground squirrels persistently infected with the hepatitis B virus-related ground squirrel hepatitis virus (GSHV) remained infected with high levels of viral surface antigen (GSHsAg) and virion-associated DNA polymerase activity in the blood for over 2 years in captivity. Unlike humans infected with hepatitis B virus, no GSHsAg-bearing ground squirrels had surface antigen in the blood without DNA polymerase-containing virions, and the levels of these did not change. Only a very mild hepatitis was observed histologically in some of the virus-infected animals, with faint Shikata-staining detectable in some. Other animals exhibited no histologic evidence of hepatitis. While the closely related woodchuck hepatitis virus has been associated with more severe chronic hepatitis and a high incidence of liver cancer in woodchucks, these were not found in GSHV-infected ground squirrels when a similar number of animals were followed for a similar time of observation. Experimental infection of ground squirrels with no evidence of current or past GSHV infection resulted in three types of response: (i) self-limited or transient GSHV-positive infection; (ii) GSHV-positive infection which became persistent, and (iii) primary anti-GSHs and anti-GSHc (antibody to the viral core antigen) responses without detectable GSHsAg or virion DNA polymerase activity in the blood. These responses are similar to those observed following experimental infection of man with HBV. GSHV produced none of the three responses when injected into a variety of laboratory animal species and a chimpanzee, causing only an antibody response to GSHsAg. No evidence of current or past infection was detected in a number of animal species from an area endemic for GSHV. The virus has only been found in one area of northern California, with a carrier rate in that location reaching 52%.