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This is a prospective case–controlled study of female attendees in Coventry. This study found an association of higher vaginal pH with chlamydial infection, independent of any other factors. Studies in vitro have shown that an acidic vaginal secretion inhibits chlamydial infection. Our objective was to analyse the association of vaginal pH and chlamydial infection in women attending a genitourinary medicine clinic. Chlamydial infections were diagnosed with ELISA and confirmed with indirect immunofluorescence. Vaginal pH was measured by a pH indicator tape ranging from 3 to 8. Consecutive female attendees with no sexually transmitted infections (STIs) were included as controls. In all, 144 female cases, diagnosed with chlamydial infection, had a median age of 20 years. Seventeen women had associated bacterial vaginosis. Eighty-two women had no other STIs. Ninety-eight women were using the oral contraceptive pill (OCP). The 145 control women had a median age of 26 years and 52 were receiving the OCP. A significantly higher vaginal pH was seen in the cases (P=0.0001, Wilcoxon test), even after adjusting for other risk factors associated with vaginal pH, including OCP use (odds ratio: 6.49, 95% confidence interval, 3.59–11.73, P=0.0001). Chlamydial infection in women was associated with a higher vaginal pH level, independent of any other factors. This study has implications for the treatment of other conditions known to lead to an increase in vaginal pH, even in asymptomatic individuals.