At SIMS XIV, we reported SIMS evidence of specific Mg2+ binding to the AT-rich regions of human metaphase chromosomes represented by G-bands. Subsequent Mg2+-depletion experiments supported a direct role for Mg2+ in promoting and maintaining the higher order chromatin structure originating G-bands, possibly due to both Mg2+-DNA and Mg2+-protein interactions. An in-depth study, reported elsewhere, implicated also Ca2+ in the maintenance of chromatin ultrastructure in the scaffold of mammalian chromosomes, in association with topoisomerase II. We examine here the association of Mg2+ with AT-rich regions of chromatin in the chromosomes of the Indian muntjac deer (IMD), leading to conclusions similar to the above. To answer the question whether the presumed divalent cation role in the chromosomes of advanced eukaryotes had an evolutionary history to be traced back to earlier evolutionary stages, we have SIMS-mapped Ca2+ and Mg2+ in BrdU-labeled polytene chromosomes from the salivary gland of the Dipteran Drosophila melanogaster. Striking Ca2+ and Mg2+ SIMS banding patterns correlating with those of the Br label (a thymidine analogue) implicate unequivocally a close association of both these cations with the AT-rich regions of DNA for these primitive eukaryotes. (c) 2006 Elsevier B.V. All rights reserved.