Human lung fibroblasts develop a cytopathic effect (CPE) when infected with Mycoplasma pneumoniae. This study was designed to determine the relationship between host cell metabolism and the formation of the CPE. Human lung fibroblasts were grown in different serum concentrations, plated at different densities, and grown for different periods of time to alter the metabolic activity of the cells. Deoxyribonucleic acid, ribonucleic acid, and protein syntheses were measured by the ability of the cells to incorporate thymidine, uridine, and leucine, respectively. With each treatment, leucine incorporation remained constant. Thymidine and uridine incorporation was higher when the cells were in high serum, at low cell densities, or grown for 2 or 3 days. The appearance of an observable CPE, which was corroborated with a protein synthesis assay, correlated closely with thymidine and uridine incorporation. A more pronounced CPE was seen when thymidine and uridine incorporation was high. In addition, it was found that the first 2 h after infection by M. pneumoniae were the critical hours in determining whether a CPE would develop. This was accomplished by altering the serum concentration of the culture medium at different times postinfection and thereby altering the metabolism of the fibroblasts. These results demonstrate the importance of the metabolic state of the host cells in studying mycoplasma infections and establish a correlation between the nucleic acid metabolism of the cells and the production of a CPE.