Growth of Chlamydia trachomatis B/TW-5/OT and L(2)/434/Bu strains in cultures of thioglycolate-activated mouse peritoneal macrophages was studied. Both strains grew to a limited extent in the macrophages, but lymphogranuloma venereum (LGV) grew better than trachoma. Growth was enhanced by centrifugation of the inoculum onto the macrophage cell layer and inhibited by pretreatment of macrophages for 2 h with 100 mug of concanavalin A per ml. No significant effect was observed by pretreatment of macrophages with diethylaminoethyl-dextran (30 mug/ml, 30 min), cytochalasin B (20 mug/ml, 1 h), and cyclophosphamide (200 mug/ml, overnight) or by treatment with hydrocortisone (1 and 10 mug/ml, overnight before inoculation and during a 2-day incubation after inoculation). Resistance to intracellular growth of the two organisms was not increased in macrophages obtained from mice immunized with the organisms compared with macrophages from nonimmunized mice unless they were pretreated in vitro with penicillin (100 U/ml, overnight). The yields of LGV organisms from the penicillin-pretreated macrophages of LGV-immunized mice were 100-fold less than from the pretreated macrophages of nonimmunized control mice. At the same time, the yields of LGV organisms from penicillin-pretreated macrophages of mice immunized with trachoma, gonococcus, and HeLa cells were not different from those obtained in pretreated macrophages of nonimmunized control mice.