Holocentric chromosomes, prepared by spreading embryo cells obtained from Megoura viciae parthenogenetic females, have been C-banded, enzymatically digested in situ using the specific endonucleases DdeI (C down arrow TNAG), DraI (TTT down arrow AAA), Tru9I (TT down arrow AA), and CfoI (GCG down arrow C), and subsequently stained with Giemsa, DAPI, CMA(3), and AgNO3. We observed that the X chromosome had the best defined banding patterns. In the M. viciae X chromosome there is a certain amount of heterogeneity in heterochromatic DNA composition. In fact, the CC-rich NOR-associated heterochromatin differs from other heterochromatic bands that are characterized by AT-rich DNAs. Our data also indicate that, in M. viciae holocentric chromosomes, all heterochromatic blocks are accessible to in situ enzyme attack, the only limit to the digestion being the presence or absence of recognition targets. This is an interesting point, since, in monocentric chromosomes, it is well known that in situ endonuclease digestion is heavily affected not only by DNA base composition but also by chromatin compactness that may limit enzyme accessibility to their specific targets.