The physical location of the mouse IFN-alpha locus (Ifa) on chromosome 4 was defined by in situ hybridization of a cloned mouse IFN-alpha probe to metaphase spreads in which one chromosome 4 was present as part of a single metacentric chromosome, all other chromosomes being acrocentric. (This approach greatly facilitates analysis and can be used even when it is difficult to obtain good banding). Using unbanded chromosomes, the grains were localized over the chromosome 4 part of the metacentric, in a region 0.61 +/- 0.07 (SD) of the distance from the centromere to the telomere. In Giemsa-banded spreads, the majority of the grains were in the region 4C3----C6. Consideration of these results and of the known linkage maps for mouse and man indicates that the Galt - Aco-1 - Ifa syntenic group spans a distance of approximately 14 cM and suggests that the same group on human 9p will also occupy a similarly sized region, with GALT proximal and IFL distal to the centromere.