Two hundred and twenty-three radiation hybrids retaining random fragments of human chromosome 16 were isolated during two successive experiments in HAT medium and screened with a total of 38 DNA probes, corresponding to anonymous DNA or gene sequences localized on chromosome 16. The presence of single or multiple human chromosomal fragments in a small subset of these hybrids was determined using in situ hybridization with total human DNA. The results confirm that individual radiation hybrids are often heterogeneous with respect to the retention and distribution of human fragments, as already suggested by their characterization with DNA probes. A number of these 223 radiation hybrids, whose detailed characterization has not been previously reported, represent a resource for the rapid isolation of new DNA markers or coding sequences from specific regions of chromosome 16 where human disease genes are already known to map.