The low-molecular-weight human 2-5A synthetase gene has been assigned to chromosome 12 using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. A cDNA probe representing almost all the coding sequences of the 2-5A synthetase gene hybridizes to four fragments of human DNA digested with the restriction enzyme EcoR1. By correlating the presence of these fragments in somatic cell hybrid DNA with the human chromosome content of the hybrids, the 2-5A synthetase gene can be mapped to chromosome 12. This contrasts with a previous assignment of this gene to chromosome 11 using an enzyme activity assay. The reason for this discrepancy remains unclear.