The chromosomes of the South American rodentCalomys musculinushave been analyzed by standard Giemsa C-banding, chromomycin A3, distamycin A and DAPI fluorochrome staining and “in situ” digestion with the restriction endonucleases Alul, HaeIII and HindIII. Constitutive heterochromatin is restricted to the centric regions, and fluoresces after either chromomycin A3 or DAPI treatments, indicating the co-existence of AT and GC-rich sequences in these regions. The restriction enzymes assayed induce diffuse, overlapping patterns of centric and interstitial bands in the chromosomes of this species. Our results agree with other authors' biochemical analyses that indicate the existence of moderately to highly repetitive DNA sequences inC. musculinusthat do not appear as a satellite in neutral CsCI DNA gradients, as well as a great abundance of the cleavage sites of these restriction enzymes in the genome of this rodent.