In constructing complete human chromosome maps, the relative order of markers and their precise chromosomal location will be combined. Multicolour in situ hybridisation, in which two probes are simultaneously hybridised to chromosomes and subsequently distinguished, potentially will provide both types of information. Using this technique, we have produced an ordered map of eight human chromosome 3 DNA markers, using small, single-copy probes that can detect target sequences ranging in size from 4 kb to as little as 500 bp.