Shifting of hepatitis A virus (HAV) epidemiology from a high towards an intermediate endemicity pattern and use of antiretroviral therapy increased the risk of HIV/HAV coinfection in developing countries. The aim of this study was to investigate the presence of HAV markers in a cohort of HIV-infected patients from 1988 to 2004. The presence of serum anti-HAV antibodies and HAV-RNA by real-time polymerase chain reaction was investigated in 581 patients. Total anti-HAV antibodies was found in 464/581 (79.8%) patients, however, achanging epidemiologic pattern of hepatitis A among HIV-infected patients from 1988 to 2004 was observed. Among patients susceptible to HAV ( n = 117), 5 (4.2%) were co-infected with HAV, all of them had IgM anti-HAV antibodies and were serum HAV-RNA-positive. The high prevalence of anti-HAV antibodies in HIV-infected patients suggests that screening tests for anti-HAV antibodies should be performed before implementation of hepatitis A vaccination, especially in those patients from endemic countries.