Backgrund: Chlamydia trachomatis is one of the most common identifiable infectious agents in neonatal conjunctivitis. It also causes pneumonitis, that is preceded by conjunctivitis in one third of cases. Aim: To asses the prevalence of Chlamydia trachomatis in newborns with conjunctivitis. Patients and methods: In 162 newborns, coming from 14 Primary Health Centers from Santiago de Chile, C. trachomatis was detected by indirect fluorescence and two polymerase chain reaction (PCR 1 and 2), wich amplified different sequences from the common endogenous plasmid. Those patients with positive indirect fluorescence and PCR 2 were definedas infected: Results: The prevalence of C. trachomatis was 8%, and the distribution of the positive cases was similar in the different Health Centers. Other isolates were: S. aureus (9.8%), S. pneumoniae (8%), S. viridans (6.2%) y H. influenzae (5.5%). Conclusions: The prevalence of C. trachomatis in neonatal conjunctivitis in Chile is similar to that of developed countries. Therefore, C. trachomatis should be considered in the election of antimicrobials for the treatment of neonatal conjunctivitis, to avoid ocular and respiratory complications. (Rev Méd Chile 2000; 128: 758-65).