By using representative strains of Chlamydia trachomatis serotypes, the efficacy of substituting fetal bovine serum with newborn calf serum for the isolation of this organism in tissue culture was examined. The criteria used were the quality and quantity of the iodine-stained cytoplasmic inclusions and the characteristics of the McCoy cells. Complete substitution of fetal bovine serum with newborn calf serum produced a detrimental change in the quality of the cytoplasmic inclusions and a dramatic decline in the inclusion count (P less than 0.001) with all of the chlamydial strains tested. There appeared to be no significant alteration in the characteristics of the McCoy cells. It is recommended from this preliminary investigation that newborn calf serum should not be used for the isolation of C. trachomatis in tissue culture.