Molecular probes were used as markers in the backcross (Czech II X BALB/cPt) X Czech II to determine the positions of six genes on mouse chromosome 16 (MMU 16). The order of the genes mapped is (centromere), protamine-1 (Prm-1), immunoglobulin lambda 1 light chain (Igl-1), preprosomatostatin (Smst), an endogenous mouse mammary tumor virus locus (Mtv-6), and two more distal sequences, superoxide dismutase, cytoplasmic form (Sod-1), and the proto-oncogene sequence Ets-2. The largest recombination frequency between any two adjacent markers is 24 cM, and thus the position of any marker on MMU 16 that is polymorphic between these two strains can be readily determined in this backcross. A region of MMU 16 which corresponds to the Down syndrome region of human chromosome 21 is located near the distal end of the chromosome.