Background The impact of a Chlamydia screening programme can be measured by the incremental amount of Chlamydia tests performed and cases detected as compared to the throughput in regular care. In the Netherlands, regular STI care is provided by specialised STI-centres (aimed at high-risk groups) and General Practitioners (GP's, basic opportunistic screening and care for symptomatic cases). An annual register-based Chlamydia screening programme is implemented in three regions since 2008. Methods The number of persons tested and cases detected in the Chlamydia Screening among 16–29 year olds in Amsterdam, Rotterdam and South Limburg, 2008–2010, were compared to consultations and diagnoses in this age group reported in surveillance data from STI centres in the regions and estimates of STI care in general practices in these regions, 2007–2010. Round 3 data are based on the first 6 months of the year. Results The baseline testing rates (at STI centers and by GP's in year pre-screening) were 10% in Rotterdam, 13% in Amsterdam and 6% in South-Limburg. CSI increased testing rates steeply in the first year to 26–30% in the cities and 17% in Limburg; this decreased to 20–21% and 13% in round 3, still doubling testing rates as compared to baseline. Positivity rates at regular STI-care facilities are higher than in CSI: 12–15% in regular care vs 4–5% in CSI; therefore the addition of CSI to case-finding in the three regions was lower than that to testing: the screening programme added about 41% on top of the cases found in regular care in round 1, but this decreased to 20% in round 3 due to lower participation and positivity rates in consecutive rounds. Conclusions By comparison to regular testing at STI centers and in general practice, the Chlamydia Screening had a major contribution towards the number of young people tested for Chlamydia in the three regions. The addition towards case-finding was lower, because the case-detection rate of the screening programme was lower than that in regular care. The Screening programme ...