Blood cells from 22 river buffaloes (Bubalus bubalisL., 2n = 50) raised in the South Italy were cultured for both early—and late—incorporation of BrdU so as to obtain G- and R-banding, respectively. Slides were sequentially treated for G-banding/Ag-NOR and R-banding/Ag-NOR techniques. These procedures allowed to identify which chromosomes carry the NORs and the frequency of active NORs per NO-chromosome. According to the standard nomenclature, NORs in river buffalo are located at the telomeres of chromosomes 3p, 4p, 6, 21, 23 and 24 while the frequencies of active NORs per NO-chromosome were higher in chromosomes 4p, 23 and 24 (64.8%, 70.2% and 72.2%, respectively) than in chromosomes 3p, 6 and 21 (42.1%, 17.3% and 28.1%, respectively).