Tumour-infiltrating lymphocytes (TIL) have been isolated from early primary melanoma (Clark level III) and expanded in vitro using culture conditions with low concentrations of IL-2 (50 U/ml). Immediately after isolation TIL consisted of mainly CD3+ T cells, and the portion of CD56+ natural killer (NK) cells was below 20%. Fresh TIL cultures could be distinguished by CD25 expression since some contained up to 33%, others less than 5% CD25+ cells. These showed differences in subsequent development during in vitro expansion. CD25-expressing cultures remained stable in their phenotype, whereas the second TIL type showed major changes: CD3 (ca 70-30%) expression decrease, CD25 (ca 5-35%) and CD56 (ca 15-55%) expression increase. The TIL type, which remained dominated by CD3+ T cells, killed autologous tumour cells efficiently (51Cr-release greater than 30% at a E/T ratio of 20:1), which could be blocked by MoAbs against MHC class I molecules. In contrast, the other TIL type exhibited weak cytotoxicity (less than 17% 51Cr-release at an E/T ratio of 20:1) against the autologous tumour. Therefore, the expression of CD25 on freshly isolated TIL is a good marker for tumour specificity of in vitro expanded TIL.