Serological responses to hepatitis B virus–X determinants have been noted in human sera, but conflicting findings concerning the correlation of anti-HBx antibodies with different stages of hepatitis B virus infection or pathological sequelae have been reported. Using an adenovirus-based eukaryotic vector, the 17 kD X protein was efficiently expressed in 293 cells. Cellular extracts containing the eukaryotic X protein have been used to screen for anti-HBx antibodies by immunoblot analysis in a large panel of sera from patients affected by hepatitis B virus chronic hepatitis, hepatocellular carcinoma and acute viral hepatitis. Sera from 32 of 171 (19%) chronic hepatitis B virus patients were positive for anti-HBx antibodies. Only one of thirty-two (3%) HBsAg-negative, anti-HBs/anti-HBc—positive chronic hepatitis serum was anti-HBx positive. Very few sera from primary hepatocellular carcinoma patients showed positivity for anti-HBx (8 of 149 or 5%). Anti-HBx were also detected in 8 of 48 (17%) acute viral hepatitis patients. In the four cases that were followed up weekly, anti-HBx anti-bodies appeared 3 to 4 wk after the onset of the clinical signs. To compare the X protein expressed in eukaryotic and prokaryotic cells as a substrate for anti-HBx antibody detection, 171 sera were screened with HBx fusion proteins expressed in Escherichia coli. The prokaryotic cell extract test seems to be more sensitive. During the chronic phase of hepatitis B virus infection, the presence of anti-HBx antibodies detected with the eukaryotic cell extract correlates with the presence of well-established markers of ongoing viral replication: serum hepatitis B virus-DNA (p < 0.001) and intrahepatic HBcAg expression (p < 0.001). (HEPATOLOGY 1991;13:143—149).