Screening polymorphic DNA probes for linkage to myotonic dystrophy (DM) and to other reported chromosome 19 (CH19) genes will develop a linkage map for human CH19. We report here the assignment of 3 cloned unique DNA sequences to 3 distinct regions of CH19. The novel use of35S-labeled probes facilitated the rapid localization of the gene for the third complement factor (C3) to 19pl3.2 by in situ hybridization. Metaphase chromosomes were from normal peripheral lymphocytes as well as from a fibroblast line containing a 15;19 translocation which permitted clear identification of CH19 regions of localization. Two random clones isolated from a plasmid library of human F-group enriched chromosomal DNA (D19S5 and D19S6) were in like manner assigned to 19pl.2 and 19ql3.2 to 19qter, respectively.