We report on the use of europium (Eu) as a suitable nonradioactive alternative for target cell labeling in limiting dilution analysis (LDA) assays set up to determine cytotoxic T-lymphocyte precursor (CTLp) frequencies. A nonradioactive alternative to the commonly used chromium-51 (51Cr) release assay seems desirable because working with radioisotopes has some major disadvantages concerning possible health risks, environmental load, costs of facilities necessary for working with radioisotopes, and shelf life. Some groups have successfully applied the Eu release assay based on detection by time-resolved fluorometry, to tests in which NK- or LAK-cell activity or cytotoxic T-lymphocyte reactions were measured. This led to the investigation whether this method could also be applicable to the more specific determination of CTLp frequencies in LDA assays. After optimal labeling conditions had been established, the sensitivity of the Eu release assay was determined by performing several LDA assays in which the target cells were labeled with either Eu or radioactive 51Cr. When CTLp frequencies were compared, it was shown that the Eu release assay is at least as sensitive and specific as the 51Cr release assay. Moreover, although the labeling procedure takes longer, sample processing is much faster: only 1 second per sample. The fact that the Eu release assay is not radioactive enables the assay to be performed at any laboratory and even--because the frequency of CTLps may have implications for organ graft survival and for donor selection in bone marrow transplantation--to do so on a routine basis.