Chlamydia pneumoniae has proved to be difficult to isolate and propagate in cell culture. We compared the growth of three strains of C. pneumoniae, TW-183 and two clinical isolates from Brooklyn, N.Y., in five cell lines, including HeLa 229, McCoy, HL, HEp-2, and HTED, an immortalized human tracheal cell line. HEp-2 was the most sensitive cell line tested. When 10-fold dilutions of three C. pneumoniae strains at known titers were inoculated into the different cell lines, the mean number of inclusion-forming units per milliliter was 1 to 2 log units higher in the HEp-2 than in the other cell lines. This difference was statistically significant. Omission of pretreatment with DEAE-dextran resulted in larger inclusions than those seen in pretreated cells, with the exception of McCoy and HTED cells. Retrieval of clinical specimens previously cultured on HeLa 229 cells and comparison of mean inclusion counts in fresh clinical specimens simultaneously inoculated on HeLa 229 and HEp-2 cells suggested that culture in HEp-2 cells may require only the initial inoculation and one passage, compared with three to four passages, as required by culture in HeLa 229 cells.