A new human myeloma cell line, ANBL-6, was established and characterized at the genotypic and phenotypic levels. The cells exhibit a clonally rearranged immunoglobulin gene locus and resemble plasma cells morphologically. The ANBL-6 cells also exhibited an absolute dependence on exogenous interleukin 6 for growth. Of interest, DNA ploidy analysis suggested the existence of a near-diploid as well as a near-tetraploid population in this cell line. Cytogenetic studies confirmed the existence of two aneuploid karyotypes and further revealed a clonal relationship between the two karyotypes, as evidenced by numerous shared structural abnormalities. To determine whether the near-diploid cells functioned as stem cells for the near-tetraploid population, the near-diploid population was separated via flow cytometry and recultured prior to ploidy analysis. This population was observed to remain predominantly near-diploid over time, suggesting that these cells did not function as stem cells for the near-tetraploid population. However, the near-tetraploid cells did exhibit a growth advantage in vitro. Moreover, sequential ploidy analysis performed retrospectively on fresh bone marrow cells from the patient also suggested that there was an expansion of the near-tetraploid population during clinical relapse. These results suggest that both populations are self-regenerating and reflect the consequences of clonal evolution in the myeloma tumor. The coexistence of clonally related subclones with shared chromosomal abnormalities, however, suggests that the near-tetraploid subclone was derived from the near-diploid subclone at an unknown time during tumorigenesis.