The finding that X antigen is associated with hepatitis B core antigen particles and that serum hepatitis B e antigen derives from the cleavage of one or more core associated polypeptides raises the question as to whether core associated X antigen could be similarly generated and released into serum. To test this hypothesis, antisera raised to X antigen peptides were used to construct an enzyme-linked solid-phase immunoassay to detect X antigen in sera from patients infected with hepatitis B virus and from woodchucks infected with woodchuck hepatitis virus. X antigen was present in more than half of the individuals tested. There was a significant association between X antigen and markers of viral replication. Most individuals destined to become surface-antigen carriers had X antigen appearing before surface antigen, as did a smaller proportion of individuals transiently positive for surface antigen. A high frequency of X antigen was observed only in sera from human populations with a high frequency of other hepatitis B virus markers. These results suggest that X is a newly identified serum marker of viral replication that often appears before surface antigen in productive infections.