The etiological diagnosis of what is today known as infection by Chlamydia trachomatis was first made possible in 1907 when Halberstaedter and von Prowazek identified inclusions in conjunctival scrapings by means of Giemsa staining. C. trachomatis was originally classified as a virus, and the culture systems used were those for viruses. Macchiavello was the first to describe the isolation of C. trachomatis in embryonated hens' eggs (1944), but the first isolation is usually credited to T'ang and co-workers (1957), also using eggs. A major step in the understanding of chlamydial infections was made in 1965 when Gordon and Quan published a paper on the use of irradiated McCoy cells for isolation purposes. This technique made it possible to perform cultures from genital specimens with simplicity in comparison to isolation from eggs. Various culture techniques have been developed parallel to the expanding knowledge of the basic biology of the genus Chlamydia. McCoy cells (mouse fibroblasts), HeLa 229 (derived from human cervical carcinoma cells) and BHK-21 cells (baby hamster kidney cells) are the cell types regularly used for the culture of C. trachomatis. The principles underlying the various culture techniques are discussed. A description of the original irradiated McCoy cell system and the simplified, sensitive technique using cycloheximide-treated McCoy cells are given in this paper.