Background Chlamydia screening programmes can only work when they motivate sufficient persons at risk to get tested regularly. The outcomes of the Chlamydia Screening Implementation (CSI) in the Netherlands are novel and valuable because of the large scale of the programme (315 000 young persons targeted), its systematic nature (invitations based on municipal registers) and its multiple screening rounds (annual invitations in three consecutive years). Methods From April 2008 to March 2010, Chlamydia Screening was offered annually to municipal-registered 16–29&emsp14;year olds in three regions of the Netherlands. A phased implementation was applied by grouping clusters in three random, risk-stratified blocks. Participation and positivity rates were compared between blocks submitted to 1, 2 or 3 screening rounds (3rd round only partially completed). The effect of the repeated screening rounds on the prevalence of Chlamydia in the whole target group was estimated by weighting procedures comparing demographic characteristics of participants and target group. Results The participation rate in the first round was 16% among all invitees and 20% among the sexually active target population (M13%, F 25%). The participation fell down to 11% in blocks invited two times and 9% in the block invited three times, whereas it was 13% in the control block invited in round two only. The positivity rate in round one was 4.2% among all participants. Positivity rates decreased significantly to 4.0% in blocks screened twice and to 3.5% in the block screened thrice (p=0.04); in the control block 4.3% tested positive. The population prevalence was estimated at 2.6% in Amsterdam, 3.8% in Rotterdam and 2.4% in South Limburg. The prevalence declined over the three screening rounds, but declines were not significant (see <cross-ref type="fig" refid="fig1">Abstract O1-S09.03 figure 1</cross-ref>). <fig loc="float" id="fig1"><no>Abstract O1-S09.03 Figure 1</no><caption> Estimated Chlamydia prevalence ...