DNA was extracted from rice plants infected with mycoplasmalike organisms (MLOs) causing yellow dwarf disease. DNA of the causal agent was separated from the host DNA by repeated bisbenzimide-CsCl equilibrium density gradient centrifugations. MLO DNA cut by HindIII was ligated into plasmid Bluescript II and cloned in Escherichia coli NM522. The DNA inserts were labeled with peroxidase and employed as probes in hybridization. Southern analysis revealed that the insert in pRYD-12 consisted of one, presumably chromosomal, piece of MLO DNA, whereas the insert in pRYD-19, another recombinant plasmid, consisted of one, presumably extrachromosomal, piece of MLO DNA. Cloned DNA probes were successfully applied in dot blot hybridization for the detection of rice yellow dwarf disease MLOs in rice plants and in an insect vector, the green rice leafhopper (Nephotettix cincticeps).