Seven Gentile di Puglia sheep (Ovis aries L., 2n = 54) reared in southern Italy were cytogenetically investigated to reveal which chromosomes carry nucleolus organizer regions (NORs). The use of sequential GBG/Ag-NOR and RBG/Ag-NOR techniques in prometaphase preparations revealed NOR-localization at the telomeres of chromosomes 1p, 2q, 3q, 6 and 25 on the basis of the standard R-banded karyotype. This procedure also permitted the characterization of G- and R-banding patterns of acrocentrics 6 and 25 which are often confused with the closest pairs in sheep and related species karyotypes. Indirect comparisons among nucleolus organizer chromosomes (NOCs) of sheep, goat, cattle and river buffalo, when using a common R-banding nomenclature, revealed that (a) sheep and goat have the same five homologous NOCs, (b) sheep and cattle have three homologous NOCs (c) sheep and river buffalo have two NOCs in common, and (d) the four species have only one homologous NOC in common.