In a preliminary study on anti-human cytomegalovirus (HCMV) antibody (Ab) by ELISA, the serum anti-HCMV/IgG Ab concentrations in 22 patients with recurrent aphthous ulcers (RAU) in the remission stage were found to be significantly higher than in 22 control subjects (121 +/- 42 vs 100 +/- 27, P < 0.05) and in 39 patients with RAU in the active stage (121 +/- 42 vs 88 +/- 45, P < 0.01). Therefore, the potential of HCMV as an etiologic agent in RAU was proposed and studies using the polymerase chain reaction (PCR) and in situ hybridization (ISH) have been performed to investigate the possible presence of HCMV DNA in pre-ulcerative oral aphthous lesions in patients with RAU or Behçet's disease (BD) of the mucocutaneous type. For this purpose, formalin-fixed biopsy specimens were obtained from 13 pre-ulcerative oral aphthous lesions, 2 samples of normal oral mucosa and 1 ileal mucosal lesion from 9 RAU patients and 4 BD patients. Five specimens of normal oral mucosa from 5 normal control subjects and 12 specimens of oral erosive or ulcerative lesions from 12 patients with erosive lichen planus (ELP) were also included. By PCR, HCMV DNA was detected in 5 of the 13 (38.5%) pre-ulcerative oral aphthous lesions, 3 from RAU patients and 2 from BD patients. The ileal mucosa specimen was also HCMV DNA-positive, whereas HCMV DNA was not demonstrated in any of the 7 specimens of normal oral mucosa from RAU patients and normal control subjects; 12 specimens of oral lesions from ELP patients were similarly negative. ISH did not detect HCMV DNA in any of the biopsy specimens from RAU patients and control subjects. Our findings suggest that HCMV may be an etiologic agent in some cases of RAU and BD.