Background Achieving adequate levels of participation and capturing high risk groups are key stones for the effectiveness of large-scale Chlamydia screening programs. We examined the determinants of (repeated) participation and Chlamydia positivity to evaluate to what extent high risk groups were reached in a large scale Internet-based screening program in the Netherlands. Methods The Chlamydia Screening Implementation was initiated in three regions among people aged 16–29&emsp14;years. Data from the first two screening rounds (2008–2010), in which approximately 280 000 persons were invited annually, were analysed. Socio-demographic and behavioural correlates of screening participation and positivity were studied in multilevel logistic regression models. Cluster was added as a second level of analysis, taking into account the effect of the neighbourhood-based invitations (to cover social or sexual networks). Results The same socio-demographic factors associated with lower screening uptake were also associated with higher Ct-positivity such as young age, non-Dutch origin, lower education, high community risk, low SES, in round 1 as well as 2. At the same time, behavioural risk factors such as having casual partners, ≥2 partners in <6&emsp14;months, concurrent partners, and a history of STI, were associated with higher participation. A small cluster effect for screening uptake was observed, independent from community risk and individual risk factors. The model for repeated participation showed that men, Turkish/Moroccans and persons ≤20&emsp14;years were less likely to participate twice, while people having a short-term relationship, a non-Dutch partner or concurrent partners were more likely to participate again. Ct-positives, who did not participate in the rescreening after 6&emsp14;months, were also less likely to participate in the second screening round. Conclusions Socio-demographic factors associated with lower participation were also associated with higher Ct-positivity, showing that, ...