The application of restriction endonucleases Alul, Ddel, Haelll, Hinfl, Mbol and Rsal in clinical cytogenetics is described. The extensive inherent heterogeneity of heterochromatin in the C-band regions revealed by these enzymes provides a valuable tool for describing the origin of trisomies and abnormal chromosomes in humans. The heteromorphic markers identified by these enzymes have tremendous potential in clinical cytogenetics. Unlike the CBG technique, slides can be stained immediately after preparation providing a rapid diagnosis. Characteristic bands induced by each enzyme in the human complement are discussed in detail. Comparative analysis of the present data, with those described earlier, revealed certain discrepancies including chromosomes 19 and 20 by Alul, chromosomes 4, 5, 8 and 22 by Mbol and chromosomes 12 and 20 by Rasl. These controversies are examined in the light of heteromorphisms, technical variables and chromosome identification.