BACKGROUND: The study assessed the acceptability of internet-based Chlamydia screening using home-testing kits among 16- to 29-year-old participants and nonparticipants in the first year of a Chlamydia Screening Implementation program in the Netherlands. METHODS: Questionnaire surveys were administered to randomly selected participants (acceptability survey) and nonparticipants (nonresponse survey) in 3 regions of the Netherlands where screening was offered. Participants received email invitations to an online survey; nonparticipants received postal questionnaires. Both surveys enquired into opinions on the screening design, reasons for (non-) participation and future willingness to be tested. RESULTS: The response rate was 63% (3499/5569) in the acceptability survey and 15% (2053/13,724) in the nonresponse survey. Primary motivation for participating in the screening was "for my health" (63%). The main reason for nonresponse given by sexually active nonparticipants was "no perceived risk of infection" (40%). Only 2% reported nonparticipation due to no internet access. Participants found the internet (93%) and home-testing (97%) advantages of the program, regardless of test results. Two-thirds of participants would test again, 92% via the screening program. Half of nonparticipants were appreciative of the program design, while about 1 in 5 did not like internet usage, home-testing, or posting samples. CONCLUSIONS: The screening method was highly acceptable to participants. Nonparticipants in this survey were generally appreciative of the program design. Both groups made informed choices about participation and surveyed low-risk nonparticipants accurately perceived their low-risk status. Although many nonparticipants were not reached by the nonresponse survey, current insights on acceptability and nonresponse are undoubtedly valuable for evaluation of the current program.