D1S1, a human anonymous DNA clone originally called lambda Ch4A-H3 or lambda H3, was mapped by two other laboratories to human chromosome 1p36 by in situ hybridization but its localization was not confirmed using a different mapping method. We used a panel of human-hamster somatic cell hybrids to show that there are copies of D1S1 on both chromosomes 1 and 3. The D1S1 clone itself is from chromosome 3, and part of it is duplicated at least twice on chromosome 1. A high frequency HindIII polymorphism detected by D1S1, believed to be at chromosome 1p36 on the basis of the in situ hybridization data, maps instead to chromosome 3. This finding demonstrates the importance of using two mapping methods to verify the localization of a gene or DNA segment, particularly a polymorphic one which itself may be used in mapping studies. It also raises the question of why in situ hybridization detected a duplicated portion of a clone but not the chromosomal origin of the clone itself.