Human hematopoietic cell lines, which had been classified on the basis of studies on clonality, and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin, and lymphoma, myeloma and leukemia lines of proven malignant origin, were tested for tumorigenic potential on subcutaneous transplantation to nude mice and for capacity to grow in semi-solid medium in vitro. Recently established LCL failed to grow both in nude mice and in agarose. In contrast, some of the LCL which had developed secondary chromosomal alterations during continuous cultivation for periods exceeding several years were tumorigenic and/or had the capacity to form colonies in agarose. Most lymphoma lines formed colonies in agarose and tumors in the mice. One of the two myeloma lines formed subcutaneous tumor which, however, showed no progressive growth. The other myeloma line failed to grow. Both myeloma lines, however, formed colonies in agarose. The myeloid leukemia line was tumorigenic while two of the three tested lymphocytic leukemia lines failed to grow in the mice. All leukemia lines formed colonies in agarose. We conclude from this study that: (1) Of the two types of Epstein-Barr virus containing cell lines [LCL and Burkitt lymphoma (BL) lines], only BL lines were shown to form tumors when inoculated subcutaneously in nude mice and had the capacity to grow in agarose in vitro. This shows that EBV transformation per se does not necessarily render lymphocytes tumorigenic in nude mice. The capacity to form colonies in agarose is not acquired either. (2) Changes of the karyotype and several phenotypic characteristics which occur in the originally diploid LCL during prolonged cultivation in vitro may be accompanied by the acquisition of the potential to grow subcutaneously in nude mice and in agarose in vitro. (3) The inconsistency with regard to the capacity of come of the neoplastic cell lines to grow in nude mice or in agarose seems to underline that neither of the two tests is a reliable criterion for malignancy of human lymphoma, leukemia and myeloma cell lines.