Cytogenetics traditionally involves the analysis of metaphase chromosomes derived from limited cell culture of cellular material. Interphase cytogenetics refers to the demonstration of any DNA sequence in interphase nuclei for the purpose of karyotypic analysis and is increasingly applied to the analysis of cells and tissues in biology and pathology. Cell culture is not required, and the technique can therefore be applied to all types of cell and tissue samples, including routinely processed, stored material. Interphase cytogenetics has contributed to the understanding of genetic organization, to the karyotypic analysis of human tumors, and to the field of prenatal diagnosis. This review comprises discussion of the history of cytogenetic analysis of the interphase nucleus, description of the probe systems used, consideration of the techniques involved, and a review of applications in the fields of biology and pathology.