We have assigned genetic markers to locations on the physical map of the genome of the archaeon Haloferax volcanii, using both a physical method (hybridization) and a more specific genetic technique (transformation with cosmids). Hybridizations were against restriction digests of each of 151 cosmids making up a minimally overlapping set and covering 96% of the genome. Results with a cloned insertion sequence and a tRNA probe indicated that transposable elements are concentrated on two of the four plasmids of this species, whereas regions complementary to tRNA are largely chromosomal. For a genetic analysis of genes involved in the biosynthesis of amino acids, purines, and pyrimidines, we used cosmid transformation to assign 139 of 243 ethyl methanesulfonate-induced auxotrophic mutations, generated and characterized for this study, to single cosmids or pairs of cosmids from the minimal set. Mutations affecting the biosynthesis of uracil, adenine, guanine, and 14 amino acids have been mapped in this way. All mutations mapped to the 2920-kilobase-pair chromosome of Hf. volcanii and seemed uniformly distributed around this circular replicon. In some cases, many mutations affecting a single pathway map to the same or overlapping cosmids, as would be expected were genes for the pathway linked. For other biosynthetic pathways, several unlinked genetic loci can be identified.