Centromeric alphoid DNAs of human chromosomes 6, 9, 16 and Y were employed to obtain information on the molecular mechanism(s) determining cytological effects produced by digestion in situ with AluI and TaqI restriction enzymes, possibly related to the structure of the above-cited areas. The following cytological and biochemical experiments were carried out using the above-mentioned alphoid sequences as probes: (1) standard in-situ hybridization and in-situ hybridization after chromosome cleavage with AluI/TaqI, and (2) filter hybridization on the DNA fractions obtained from the material solubilized and that retained on the slides after digestion in situ with AluI/TaqI. Biochemical data show that cleavage of alphoid DNAs is not prevented by the peculiar organization of centromeric heterochromatin, but such cleavage is not necessarily followed by complete DNA solubilization. The analysis of alphoid sequence cleavage in naked genomic DNA as well as during digestion of fixed chromosomes shows that (1) AluI cuts more efficiently than TaqI, (2) DNA fragments as large as 3-5 kb can be solubilized, and (3) DNA fragments of the same size are found in both fractions of DNA, i.e. that retained on the chromosomes as well as that solubilized from chromosomes. Cytological data show that previous chromosome digestion, mostly with TaqI, increases the hybridization signal area, suggesting that this fact might be due to (1) chromatin reorganization produced by enzyme attack and/or (2) the presence of alphoid DNAs which might be restricted not only to the kinetochore area but also to para/peri-centromeric heterochromatin. Lastly, centromere DNA solubilization as a consequence of restriction enzyme cleavage seems to vary from chromosome to chromosome, thus suggesting that centromeric regions do not represent a homogeneous class of constitutive heterochromatin.