The aim of this study was to determine the frequency and significance of the tumor DNA content heterogeneity in 33 previously untreated human neuroblastomas. We used image cytometry to selectively analyze neuroblasts by excluding karyorrhectic or stromal cells from cytometric measurements. DNA content heterogeneity with more than one clonal subpopulation on DNA histogram was found in 8 of 33 cases. Of these 8 cases, 4 showed MYCN amplification. Double labeling fluorescent in situ hybridization with probes for the centromeric region of chromosome 2 and MYCN gene was used to confirm the DNA content heterogeneity. DNA content heterogeneity was associated with poorer prognosis in this study (P<0.05). There was a significant correlation between euploidy (di- and tetraploidy) and worse prognosis, but only when heterogeneous neuroblastomas with euploid cell population were assigned to euploid tumors (P=0.006). Our results may explain the conflicting data in the literature regarding ploidy and suggest that DNA content heterogeneity and the presence of a euploid population may predict worse prognosis in neuroblastoma patients.