A number of parameters aimed at optimizing culture conditions for both Sf1Ep cells and Treponema pallidum have been investigated. Optimum temperature for replication of T. pallidum ranged between 33 and 35 degrees C. At 33 degrees C, replication occurred in the presence of atmospheric oxygen concentrations of less than 0.3 to 10%, the optimum range being 1.5 to 5%. No replication occurred in the presence of 12.5% oxygen. When both temperature and oxygen concentrations were varied between 33 and 35 degrees C and 1.5 to 5%, respectively, little differences in replication were noted. Although variation in the oxygen concentration within each temperature group had little or no effect on replication, it did have an effect on motility, which remained greater in the 5% oxygen concentration after 9 to 12 days of cultivation. Optimum concentration of fetal bovine serum in the culture medium was 20%, although replication occurred in concentrations ranging from 5 to 30%. If carefully screened, calf serum could be substituted for fetal bovine serum. Testis extract was an essential component of the culture medium. Although extract obtained from an adult rabbit--either normal or T. pallidum infected--was slightly superior, replication of T. pallidum occurred when rat or hamster testis extract was substituted.