To assess the pathogenetic role of Chlamydia trachomatis in non-bacterial prostatitis (NBP), aspiration biopsied specimens were examined for C. trachomatis by using in situ DNA hybridization and antibody titer to C. trachomatis was measured. An enzyme-linked immunosorbent assay (ELISA) for C. trachomatis specific IgA was employed using purified C. trachomatis type L2 EBs. The positive rates of IgA antibodies to C. trachomatis in serum, EPS and VB3 were 25.6%, 31.5% and 29.4%, respectively. They were significantly higher (p less than 0.05) in the control groups. A good correlation (0.78) of IgA antibody titer to C. trachomatis was found between EPS and VB3. In 9 husbands with positive antibodies to C. trachomatis, 5 wives showed positive serum antibodies. In the NBP patients with a high positive antibody titer, the decrease of titers was shown after treatment with drugs effective against C. trachomatis. Transrectal aspiration biopsies were performed on 7 patients with high positive IgA antibody titers to C. trachomatis, and 2 specimens showed hybrids in the cells by using in situ DNA hybridization. These findings indicate that C. trachomatis is a predominant pathogen for NBP.