By co-cultivating peripheral blood lymphocytes from normal male donors with cells from a line designated Lma-66VP. established from a female donor and si- multaneously producing both HTLV-I and EBV, 2 continuous culture lines were obtained. Normal male lymphocytes were considered to be immortalized by co-cultivation because they were of male karyotype and lacked the 3q+ chromosome that was observed in all Lma-66VP cells. These immortalized cultures were designated Co-culture I and Co-culture 2. After prolonged cultivation, Chromosome abnormalities characteristic of each co-culture line were observed in all cells examined, indicating their clonal origin. Immortalized cells initially produced both HTLV-I and EBV. Although production of EBV was not seen in the co-culture lines after prolonged cultivation when all cells examined had the characteristic chromosomal abnormality indicating their clonality, EBNA+ cells persisted. Thus, clonal cells were doubly infected by HTLV-I and EBV. While the majority of clonal cells in Lma-66VP. Co-culture I, and Co-culture 2 lines expressed a surface marker of 6-cells, small numbers of cells expressed a T-cell surface marker. These findings demonstrate that human lymphocytes immortalized following dual infection by HTLV-I and EBV exhibit chromosome rearrangement, resulting in expression of surface markers inconsistent with their differentiation lineage.