With the advent of nonradioactive labels, in situ hybridization (ISH) has become a useful technique for the detection of viral DNA in infected tissue ( 1 ), mRNA expression ( 2 ), sex determination ( 3 ), human gene mapping ( 4 ), and interphase cytogenetics ( 5 – 8 ). For chromosomal mapping of genes by ISH, labeled DNA probe is hybridized to metaphase spreads. If the label is a radioisotope, the signal is detected by autoradiography ( 9 ). Nonradioactive labels, such as biotin ( 4 ), digoxigenin ( 10 ), or 2-acetylaminofluorene (AAF), are detected by immunocytochemistry. AAF is incorporated by chemical modification of the DNA probe. Biotin and digoxigenin are incorporated enzymatically by nick translation or by random primer extension ( 11 ), although both can be incorporated by chemical modification ( 12 ). In the authors’ laboratory, biotin has been successfully used in mapping genes with probes of 0.8 Kb ( 10 ). The method described here, therefore, applies to biotin-labeled probes although, with minor modifications, can be adapted to probes labeled by digoxigenin or AAF. An example of chromosomal mapping of a unique sequence is shown in Fig. 1 . Fig. 1.  Human chromosome spread probed with a biotdnylated β-globin probe. There is a signal at band pl.5 (arrow) on chromosome 11.