A novel virus-like infectious agent (VLIA), obtained by direct transfection of DNA from Kaposi's sarcoma of a patient with acquired immune deficiency syndrome (AIDS), was transmissible from culture to culture by cell-free filtrate. VLIA contained an outer limiting membrane and had a buoyant density of 1.17–1.20 g/ml in a sucrose gradient. The DNA genome of VLIA was estimated to be >150 kilobase (kb) pairs and carried repetitive sequences. An 8.6 kb pair cloned probe (psb-8.6) and a 2.2 kb pair cloned probe (psb-2.2) of VLIA detected specific sequences in DNA of VLIA infected cells, but not in DNA of uninfected NIH/3T3 cells. By Southern blot hybridization analysis, VLIA was distinct from all known members of human herpes virus, from vaccinia virus, monkey herpes virus saimiri (HVS), and mouse cytomegalovirus (MCMV). Using synthetic primers with the VLIA specific DNA sequences and the polymerase chain reaction (PCR) method, we detected VLIA sequences in DNA isolated from 7 out of 10 patients with AIDS. VLIA infection was identified in spleen, liver, brain, lymph node, Kaposi's sarcoma tissues, or peripheral blood mononuclear cells from these patients, but not in 5 different organs and a tumor from 5 subjects without AIDS. Antiserum raised against VLIA in rabbit positively immunostained brain and lymph node tissues from these AIDS patients.