Somatic and germ cells of the gray short-tailed opossum (Monodelphis domestica) were analyzed cytogenetically by C-banding, G-banding, and silver staining. The diploid chromosome number was found to be 18. C-banding revealed constitutive heterochromatin at the centromeres of all chromosomes, with additional heterochromatin on one arm of the Y. Silver staining for the localization of nucleolus organizer regions (NORs) showed NORs on the X and on a pair of autosomes, tentatively designated as chromosome 5. In females, the NORs on both X chromosomes were active in 98.3% of all somatic cells analyzed, indicating that this locus escapes sex-chromosome dosage compensation. Chromosome 5 showed an active NOR in only 16.2% of cells scored from the six females. In males, 100% of the somatic cells analyzed showed an active NOR on the X chromosome. Greater variability of autosomal NOR activity was observed between male individuals and between different tissues of the same male. Silver staining of pachytene spermatocytes revealed that (1) only an autosomal NOR was active, whereas the NOR of the X chromosome was inactive, and (2) the X and Y chromosomes showed asynaptic behavior, even though all autosomal synaptonemal complexes were fully formed during this stage.