Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.