The success of large-scale screening for Chlamydia trachomatis depends on the availability of noninvasive samples, low costs, and high-quality testing. To evaluate C. trachomatis testing with pregnant women, first-void urine specimens from 750 consecutive asymptomatic pregnant women from the Rotterdam area (The Netherlands) were collected. Initially, we investigated the performance of three different DNA isolation methods with 350 of these urines and 70 pools of 5 of the same subset of urine samples. The routinely used COBAS AMPLICOR test was compared to the COBAS AMPLICOR test with prior DNA isolation by use of the MagNA Pure large-volume kit and the MagNA Pure bacterial DNA isolation kit. The latter combination provided the best DNA test for pooled urines, with a sensitivity twice that of the other methods. Next, using all 750 urines, the COBAS AMPLICOR performance for individual testing was compared to pooled testing with the standard COBAS AMPLICOR procedure and subsequently to pooled testing with COBAS AMPLICOR in combination with the MagNA Pure bacterial DNA isolation kit. The sensitivity of COBAS AMPLICOR was 65% on individual and 42% on pooled urines but improved to 92% on pooled urines with the MagNA Pure bacterial DNA isolation kit, making this combination the best screening method. The C. trachomatis prevalence in this population appeared to be 6.4%. Additionally, the cost of the combined MagNA Pure bacterial DNA isolation kit and COBAS AMPLICOR method on pooled urines was only 56% of the cost of the standard COBAS AMPLICOR test applied to individual urines. Costs per positive case detected in the combined method were 39% of standard costs.Chlamydia trachomatis is one of the major sexually transmitted pathogens, and high prevalences of chlamydial infection have been documented for asymptomatic women in many European countries (34). Asymptomatic carriers are of substantial importance in the transmission of C. trachomatis infection within a community. Asymptomatic chlamydial infection in pregnant women imposes an additional risk for acute and chronic consequences for the women themselves and their (unborn) offspring (2,6,8,9,16). In The Netherlands, C. trachomatis causes most sexually transmitted infections, with approximately 60,000 new cases estimated for a total population of 16 million in the year 2000. Studies in general practice have shown an increase in the incidence of chlamydial infections (26), but data covering other specific target groups outside of the sexually transmitted disease (STD) outpatient clinics are sparse (1). Dutch population-based screening for C. trachomatis is still under debate, with cost-effectiveness of screening, complexity of sampling, the reliability of test methods, and the nature of the target population as major issues of discussion (23).In order to investigate the prevalence of chlamydial infection during pregnancy in Rotterdam, and the risk factors and consequences of chlamydial infection during pregnancy for women and newborns, a follow-up study was planned. We ex...