Despite the likely prevalence and documented biological impact of inverted DNA sequences in humans and other species, our ability to detect them on a routine basis is limited. The technique of chromosome orientation fluorescence in situ hybridization (CO-FISH) was used to detect obligate chromosome inversions associated with isochromosome formation in two human cell lines. Simultaneous hybridization of a strand-specific telomeric probe allowed us to deduce the absolute orientation of repetitive DNA sequences associated with the inverted region. These results show that, in principle, CO-FISH could be used to detect virtually any type of inversion, including those likely to escape detection by other methods. Prospective applications of the technique are discussed in relation to its principal limitation, the present availability of suitable single-stranded DNA probes.