To determine differences in biological features among different adrenal tumors, we investigated the DNA ploidy, numerical chromosomal aberration and proliferative activity in human adrenal cortical neoplasms. Our study included six adrenal cortical adenomas with Cushing syndrome, 12 adenomas with hyperaldosteronism, three non-functioning adenomas and three adrenal cortical carcinomas. Isolated nuclei from frozen samples were used for fluorescence in situ hybridization (FISH) analysis, and formalin-fixed, paraffin-embedded tissues from the same materials were analyzed using flow cytometry (FCM) for DNA ploidy. Sections from paraffin blocks were stained immunohistochemically with antibodies against Ki-67 and p53. For FISH analysis, we used an alpha-centromeric enumeration probe for chromosome 17. The mean Ki-67 labeling index (LI) of adrenal cortical carcinomas was markedly higher than that of adrenal cortical adenomas (209.4 vs 8.7). In functional adrenal cortical adenomas, the LI was significantly lower in adenomas with hyperaldosteronism than in those with Cushing syndrome (P = 0.004), although FCM results indicated that tetraploid patterns were more frequently observed in the former type. Tumor size was significantly smaller in adenomas with hyperaldosteronism than in those with Cushing syndrome (P = 0.004). Chromosome 17 showed disomy in all adrenal cortical adenomas, whereas chromosome 17 abnormalities were found in two of three adrenal cortical carcinomas. Only the latter two cases strongly expressed p53 protein. Our study characterized various biological features of benign and malignant adrenal cortical tumors. The use of a combination of markers might provide additional information to assist our understanding of the clinical behavior of an individual adrenal cortical tumor.