Abstract Background ACCEPt, a large cluster randomized control trial, aims to determine if annual testing for 16 to 29 year olds in general practice can reduce chlamydia prevalence. ACCEPt is the first trial investigating the potential role of practice nurses (PN) in chlamydia testing. To inform the design of the ACCEPt intervention, we aimed to determine the chlamydia knowledge, attitudes, and testing practices of participating general practitioners (GPs) and PNs. Methods GPs and PNs from 143 clinics recruited from 52 areas in 4 Australian states were asked to complete a survey at time of recruitment. Responses of PNs and GPs were compared using conditional logistic regression to account for possible intra cluster correlation within clinics. Results Of the PNs and GPs enrolled in ACCEPt, 81% and 72% completed the questionnaire respectively. Less than a third of PNs (23%) and GPs (32%) correctly identified the two age groups with highest infection rates in women and only 16% vs 17% the correct age groups in men. More PNs than GPs would offer testing opportunistically to asymptomatic patients aged ≤25 years; women having a pap smear (84% vs 55%, P