The human 5S rRNA genes have been localized by in situ hybridization to metaphase chromosomes. Tritiated RNA probes were made by transcription from 2,300-bp and 638-bp DNA fragments containing an isolated human 5S rRNA gene. Hybridization to metaphase spreads from a balanced reciprocal translocation carrier, 46, XX, t(1;7)(q42.13;p11.1), showed that the 5S rRNA genes were entirely localized on the normal and the derivative chromosome 1. This narrows the chromosome position of the major fraction of 5S rRNA genes and pseudogenes to the region 1q42.11→q42.1