We and others previously reported DNA polymerase activity in culture supernatants of peripheral blood mononuclear cells from patients with acute Kawasaki syndrome (KS). In the present study, we further characterized the previously detected polymerase activity and attempted to confirm its presence in cultured peripheral blood mononuclear cells from additional patients with KS. Characterization experiments indicated that the polymerase activity was typical of a DNA-dependent DNA polymerase rather than viral reverse transcriptase. Peripheral blood mononuclear cell cultures from 17 additional KS patients were negative for reverse transcriptase activity in three laboratories. Our findings do not provide support for a retroviral etiology of KS. Further studies should continue to focus on infectious agents in efforts to elucidate the etiology of KS.