Primed in situ labeling (PRINS) is a sensitive and specific method that can be used for the localization of single copy genes and sequences too small for detection by conventional fluorescence in situ hybridization. By the use of PRINS, the human SRY gene was localized to Yp11.31-p11.32 and the SOX3 gene to Xq26-q27. In other studies, we localized specific deletions of RBM and DAZ, candidate genes for AZF (azoospermia factor) to proximal Yq11.2, the AZF region, in an infertile male. Locus-specific oligonucleotide probes (PRINS primers) were annealed to chromosomal DNA in situ and extended on preparations fixed on glass slides in the presence of dATP, dCTP, dGTP, dTTP, biotin-16-dUTP, and Taq DNA polymerase. After addition of avidin-conjugated fluorophore, signals were visualized by fluorescence microscopy in metaphase spreads from patients and controls. With further development, the PRINS method may prove useful for localization of single-copy genes, in general, and for the detection of gene deletions.