Restriction analyses and DNA/DNA hybridisation of parasite DNA isolated from monkeys infected with the malarial parasite Plasmodium knowlesi has permitted unambiguous identification of the nuclear DNA of this species. Its (G+C) content, as determined by estimations of buoyant density as well as by direct analysis, is about 38%, essentially indistinguishable from that of its primate laboratory host, and grossly different from that of the major human malaria parasite, P. falciparum, which has a (G+C) content of approx. 19%. In addition, gradient fractionation of total P. knowlesi DNA revealed a minor DNA component (approx. 1% of the total) with a (G+C) content of about 19%. This DNA comprises covalently closed circular molecules which have a contour length about 11.6 microns, carry a small cruciform structure, and are thought to originate in the parasite's mitochondria.