The clinically important issue of tumor heterogeneity was studied in c5mL/G-Ep-myc transgenic mice, which provide a genetically uniform model system in which all animals eventually develop B cell lymphomas after additional genetic changes beyond enforced expression of the transgenic oncogene. Three different approaches were compared for discerning the cellular and genetic homogeneity of these tumors. Analysis of Igh gene rearrangement showed mainly monoclonality and only infrequent oligoclonality in the tumors from a given animal. In contrast, cytogenetic examination indicated a substantial degree of heterogeneity in the tumors from a given animal and showed that a wide variety of secondary genetic changes occur in E p - myc transgenic mice. Flow cytometry of DNA content also revealed a high degree of heterogeneity within and among the tumor masses from single Ep-myc mice. Estimates of tumor heterogeneity revealed by these three techniques often did not coincide, indicating that these different approaches reflect distinct cellular parameters. Transgenic Ep- myc mice additionally homozygous for the scid mutation displayed enhanced levels of secondary genetic changes that were valuable for the methodological comparisons performed here, and demonstrated that the extent of tumor heterogeneity can be influenced by specific genes other than the primary Ep-myc transgene. In summary, a combination of methodologies appears to be required to reveal the full extent of tumor heterogeneity within a single individual. 0 1996 Wiey-Liss, ~n c .