One line of investigation of cellular events leading to rejection of an allograft has been to collect the cells infiltrating the rejected allograft and to subject them to in vitro functional and cell marker analysis. Outlined in this article is a description of the limiting dilution analysis technique applied to mechanically disrupted cells obtained from three different rejected human kidney allografts and a phenotypic cell surface marker, as well as a functional study of the progeny of such cells at a clonal level. Of the harvested cells, 65% were T11+, 58% were OKT8+, and 14% were OKT4+. The frequencies of colony forming cells (CFC) were assessed in liquid medium supplemented with lectin-free purified IL-2 (selecting in vivo activated cells) or with IL-2 plus PHA. The CFC frequencies ranged from 1/777 to 1/120 cells plated and from 1/250 to 1/90 cells plated in, respectively, IL-2 and IL-2 plus PHA. Only colonies with a probability of monoclonality more than 80% were further expanded in the presence of lectin-free IL-2. Among 31 colonies grown in the presence of IL-2 alone, all colony-cells were OKT11+, 4/31 were T4+T8-, 24/31 T4-T8+--and, finally, 3/31 were T4+T8+. On the other hand, 122 colonies grown in the presence of IL-2 plus PHA were also all OKT11+, but 47/122 were T4+T8-, and 62/122 were T4-T8+. In addition, expression of DR molecules was highly variable from colony to colony. Significant antidonor cytotoxicity was recorded in 24 colonies, most of them expressing T8 molecules at their surface. Moreover cytotoxic antidonor colonies seemed to recognize an antigen determinant different from the known HLA incompatibilities between donor and recipient. In three long-term-cultured colonies, we noticed a shift in surface marker: from the expression of T8 to either the coexpression of T4 or the loss of the T8 to the sole expression of the T4 molecules. This methodology is a step forward in the elaboration of techniques for determining the relationships between the surface marker identity and the immune function of cells activated in vivo, which are found in a rejected human kidney.