Chlamydial conjunctivitis is a disease associated with venereal transmission through direct sexual contact or autoinoculation with genital secretions. Appropriate therapy for patients and their sexual partners involves important questions regarding the source of infection and mode of transmission. This study explored the potential role of a fomite, i.e., an environmental surface, as a possible vector of transmission. We determined the in vitro recovery of Chlamydia trachomatis from a nonporous plastic surface under ambient and humid conditions using the standard shell vial technique and confirmation by direct monoclonal immunofluorescence. Under ambient conditions, the TP50 (time at which 50% of samples were positive for Chlamydia) was 5 min, with complete desiccation occurring at 45 min. Under humid conditions, the TP50 was 52.5 min and complete desiccation did not occur up to 3 h. Beyond 45 min, a significantly greater number of positive chlamydial samples were collected under humid conditions (11 of 30) than under ambient conditions (0 of 30) (p = 0.00016). We conclude that a fomite, such as a nonporous plastic surface, may serve as a potential vector for the transmission of chlamydial infection to the eye, especially under humid conditions. This new information may prove useful in counseling patients and their sexual partners.