The nature of the serum requirement of Treponema pallidum subspecies pallidum (Nichols strain) was examined in a culture system utilizing Sf1Ep cottontail rabbit cells. In this system, significant multiplication of treponemes occurs in the presence of select lots of fetal bovine serum (FBS) or calf serum (CS) at concentrations of greater than or equal to 5% (vol/vol). Heat-inactivation of the serum greatly enhances treponemal multiplication, and normal human serum was found to be as effective as FBS in supporting the growth of T. pallidum. The protein fraction of FBS obtained by membrane ultrafiltration was capable of supporting the multiplication of T. pallidum when added to the basal tissue culture medium; an average increase of 23-fold was observed in these cultures, as compared with a mean increase of 25-fold in the 20% FBS controls. In contrast, the ultrafiltrate fraction of FBS (consisting of compounds with molecular weights of less than 10,000 daltons) did not support either growth or the retention of motility. Proteins precipitable with 25% (wt/vol) polyethylene glycol (i.e., albumin, transferrin, ceruloplasmin, and other proteins) also promoted the growth of T. pallidum. This observation provides further evidence that the required serum components are associated with the protein fraction.