The aims were 1) to estimate the prevalence of C. trachomatis infection among sexually active female students in Kaunas, Lithuania; 2) to investigate the usefulness of personal invitation, self-sampling, and pooling of samples for screening; and 3) to evaluate the costs of the approaches used. A cross-sectional study inviting 795 female students (18-31 y of age) from 7 high schools and 1 college in Kaunas was performed. The response rate was 67% (533/795). Self-obtained vaginal samples were analysed, individually and pooled (n = 3), using Digene Hybrid Capture II CT/NG Test. The overall prevalence of C. trachomatis infection was 5.6%. Among the sexually active female students 20-24 y of age (n = 424), the prevalence was 7.1%; however, the prevalence varied from 0% to 14.2% at the different schools. For estimation of the population prevalence based solely on identification of C. trachomatis positive pools, the pooling strategy reduced the costs by 85%. For estimation of population prevalence and for diagnosis of each individual sample, pooling reduced the costs by 70%. Targeted screening, using pooling to reduce the expenses, mainly of 3rd and 4th y Lithuanian female students could be recommended. By extended personal contact and internet-based communication, increased participation rates may be attained.