This study sought to investigate trends in the prevalence of genital Chlamydia trachomatis infection in heterosexual patients attending an urban sexual health service. Data from cases of C. trachomatis in all new self-referred heterosexual patients who were tested at the Sydney Sexual Health Centre from 1994 to 2000 were extracted from the Centre's database. Female sex workers and homosexually active men were excluded. Over the study period the prevalence of C. trachomatis infection doubled from 1.8 per cent to 3.5 per cent among the women (p=0.004) and tripled from 2.1 per cent to 6.6 per cent among the men (p<0.001) who were tested. Both men and women reported an increasing overall trend in the mean (but not median) number of sexual partners during the previous 3 months (p=0.039 and p=0.001, respectively). There were modest increases in the proportion of men and women that reported unprotected vaginal or anal sex in the previous 3 months, from 76.5 per cent to 81.7 per cent for males (p=0.122) and from 65.1 per cent to 70.2 per cent (p=0.01) for females. The introduction of more sensitive DNA-based testing probably only accounted for 8 per cent of the rise in prevalence among women and 16 per cent among men. These findings complement the rising trends in national notifications of C. trachomatis infection. Further investigation and interventions on a national scale to reduce the prevalence of C. trachomatis seem timely.