The helper T lymphocyte precursor (HTLp) frequency detected in HLA matched sibling donors is directed against alloantigens and this frequency has been shown to correlate with the development of graftversus-host disease in the patient after bone marrow transplantation. Using limiting dilution analysis the HTLp frequency is determined by measuring IL-2 using the IL-2-dependent murine cell line CTLL-2. An important factor associated with the successful performance of any bioassay is the reliability of the indicator cell line used. When maintained in continuous culture, however, the overall sensitivity of this cell line is highly variable. To overcome this problem, large batches of CTLL-2 were grown, frozen and tested for their ability to detect low levels of IL-2 (0.01 IU) in dose-response assays. Batches meeting this criterion were stored in liquid nitrogen until required. In this study, we investigated the effect of freezing and storing large batches of this cell line, thereby assuring a plentiful supply of sensitive indicator cells.