In this study, 5-iodo-2'-deoxyuridine-, cytochalasin B-, and cycloheximide-treated and untreated McCoy cells were inoculated with representative strains of Chlamydia trachomatis serotypes, as well as with clinical specimens from a pediatric population, and the quality and quantity of the cytoplasmic inclusions were examined. Cycloheximide treatment consistently rendered the highest number of quality inclusions per milliliter of any method tested for both the laboratory strains (P less than 0.001) and the clinical isolates (P less than 0.01). The inclusion counts in untreated McCoy cells were either higher than or not significantly different from those in 5-iodo-2'-deoxyuridine- an cytochalasin B-treated cells. However, the quality of the inclusions, using the criteria of size, brightness, and ease of detection produced in untreated and 5-iodo-2'-deoxyuridine-treated cells, was inferior to the quality of those obtained by other procedures. Cells treated with cytochalasin B had the best-quality inclusions as compared with those treated by any other method. It is recommended from our experience that for overall sensitivity and simplicity cycloheximide treatment is the method of choice for isolation of C. trachomatis in McCoy cells.