Eleven mammalian cell lines, HeLa 229, HeLa M, Hep-2, FT, BHK-21, Vero, MK-2, MPK, L-WO5A2, McCoy, and L-929, were tested for their susceptibility to infection with Trachoma strains TW-3 (type C, ocular origin) and UW-5 (type E, genital origin). All the cell layers were pretreated with diethylaminoethyl-dextran before inoculation of the organisms, and the inocula were centrifuged onto the cell layers. HeLa 229 was found to be the most sensitive to infection as determined by inclusion counts. The next most susceptible were cell lines MK-2, Hep-2, McCoy, and HeLa M, in that order. Infectivity in these cells ranged from 89 to 12% of that observed in HeLa 229. The remaining cell lines, BHK-21, L-WO5A2, L-929, Vero, MPK, and FT, were much less susceptible with infectivity less than 10% that of HeLa 229. HeLa 229 cells and 5-iodo-2'-deoxyuridine-pretreated McCoy cells have been used most extensively with Trachoma organisms in our laboratories. Infectivity in these two cell culture systems, both pretreated with diethylaminoethyl-dextran, was compared using 13 Trachoma strains of both ocular and genital origins of different immunotypes. The two systems performed similarly except with two type C, three type I, and one type J strains, With the type C, I and J strains tested, considerably fewer inclusions were found in 5-iodo-2'-deoxyuridine-pretreated McCoy than in HeLa 229 because inclusion formation of these strains in McCoy cells was not enhanced by 5-iodo-2'-deoxyuridine pretreatment.