Differentiation between well-differentiated hepatocellular carcinoma (HCC) and nonmalignant lesions with increased cellular proliferation may be difficult in needle biopsies. Based on recurrent chromosome aberrations known for HCC, we developed a nonfluorescent in situ hybridization technique that allows combination with morphological analysis in bright-field microscopy. Fourteen biopsies of HCC and 31 samples of regenerative nodules (n ‫؍‬ 10), chronic hepatitis (n ‫؍‬ 10), fibrosis or cirrhosis of unknown origin (n ‫؍‬ 5), focal nodular hyperplasia (n ‫؍‬ 2), primary biliary cirrhosis (n ‫؍‬ 2), steatosis (n ‫؍‬ 1), and adenomatous hyperplasia (n ‫؍‬ 1) were analyzed with probes specific for the centromeric regions of chromosomes 1, 6, 7, and 8. After microwave pretreatment and in situ hybridization, signals were detected using a tyraminebased system and AEC as substrate. Evaluation of signals was done by conventional bright-field microscopy. Using this approach, aberrant counts were seen for at least one chromosome in 12/14 cases of HCC. In contrast, none of the nonmalignant lesions revealed aberrant counts for any of the chromosomes analyzed. In conclusion, this new combination of in situ hybridization and tyramine amplification allows fast and reliable evaluation of chromosome aberrations in a histomorphological context similar to paraffin immunohistochemistry. Registration of imbalances contributes to a reliable differentiation between malignant and nonmalignant lesions of the liver. The fast and reliable differentiation between malignant and nonmalignant tumorlike lesions of the liver is of the utmost importance for the further treatment and surgical procedure of the patients. However, even for the experienced pathologist, in some cases, determining the correct diagnosis may be extremely difficult and uncertain, particularly if only small biopsies are available as the main source for histological examination. In particular, differentiation between well-differentiated hepatocellular carcinoma (HCC) and benign alterations may be impossible based on morphologic criteria alone (1). Detection of chromosomal aberrations within questionable tumors could contribute toward solving this problem. Because of the time and effort required for conventional cytogenetics, this technique is usually not appropriate. The recently developed comparative genomic hybridization (CGH) has revealed promising results (2,4,11,13,14), but it is also of limited value because of the duration and amount of tissue required. By contrast, fluorescence in situ hybridization (FISH) yields results that are evaluable by simple counting of fluorescence signals in conventional biopsies (12). A major limitation of this approach is, however, that most pathologists are not familiar with the morphology of the tumor in histological sections counterstained with fluorescent dyes. Furthermore, many histomorphological details remain undetected in the dark field required for evaluation. In addition, epifluorescence microscopy is based on expensive technica...