The centromeric regions and alpha-satellite I sequence were studied on chromosomes 1, 29 and the rob(1;29) translocation in a Portuguese breed of cattle, Barrosa, carrying the translocation. Rob(1;29) centromeric regions showed heterochromatic bands with propidium iodide but, unlike the acrocentric autosomes, no strong centromeric bands were revealed with chromomycin A3. An alpha-satellite I sequence was not found at the centromeres of the X, Y and rob(1;29) chromosomes in the breed, although it was present at the centromeres of all acrocentric chromosomes including 1 and 29. Restriction enzyme banding with MspI revealed polymorphisms between different rob(1;29) chromosomes in both centromeric and intercalary regions. The data show that the centromeric region of the rob(1;29) chromosome has lost the alpha-satellite I sequences, while retaining other heterochromatin, and suggest that this important and widespread translocation has occurred multiple times.