It has earlier been shown by in situ hybridization that the 5 S RNA genes are located in region 2A of chromosome II in chironomus tentans [1]. In the present study the resolution in this chromosome region could be considerably improved by using the inversion 1A/9C of chromosome II, which carries region 2A, not in the usual position close to one end of chromosome II, but in the middle of the chromosome. It was then revealed that electrophoretically isolated 5 S RNA hybridized in situ with two adjacent bands in the region 2A. It was also observed that in heterozygotes harbouring one normal chromosome II and one with the inversion 1A/9C, there is a close physical contact between the bands containing the 5 S RNA genes and the nucleolar organizer region.