SummaryPropagation of the transplantable acute rat leukemia L5222 in vitro was highly dependent on the reducing agent 2-mercaptoethanol (2-Me). Maximum growth promotion was obtained in the simultaneous presence of 1.25–2.5×10⁻⁴ m 2-Me and 30% fetal bovine serum in enriched tissue culture medium NCTC 135. A short exposure to the reducing agent insured growth of the leukemia cells for 5 days, but extended culture necessitated constant presence of the thiol. After 2–8 days of culture, inoculation of the cell suspension produced widespread leukemia in inbred BD IX rats.