The region of human chromosome 19 which includes the myotonic dystrophy locus (DM) has recently been redefined by the tight linkage between it and the gene for muscle-specific creatine kinase (CKMM), which lies just proximal to DM. Utilizing human/hamster hybrid cell lines containing defined breakpoints within this region, we have assigned a number of new probes close to DM. Two of these probes, p134B and p134C, were isolated from a single cosmid clone (D19S51) and detect the same BglI RFLP; p134C detects an additional RFLP with the enzyme PstI. Analysis of these probes in the Centre d'Etude du Polymorphisme Humain families demonstrates tight linkage with a number of markers known to be proximal to DM. A two-point lod score of 6.34 at theta = .025 demonstrates the linkage of this probe to DM. Analysis of a DM individual previously shown to be recombinant for other tightly linked markers indicates that p134C is distal to DM. This result indicates that both the new probe and the existing group of proximal probes including CKMM and ERCC1 probably flank DM and define the genetic interval into which this mutation maps.