The aim of this investigation is to make in-depth comparative studies at the chromosomal, protein and DNA levels among members of the cat family. However, the phylogeny of the cat family (Felidae) is uncertain. Mitochondrial DNA (mtDNA) has been chosen as an evolutionary probe. The molecule evolves rapidly, is maternally inherited and provides a high degree of resolution among closely related species. mtDNA has been isolated from the domestic cat (ten individuals), lion, cheetah (three individuals), and clouded leopard (two individuals). In addition to the four aforementioned species, mtDNA has been isolated from a cougar and a leopard. The mtDNAs have been digested with 17 different restriction enzymes and cleavage maps have been constructed for four of them (lion, cheetah, domestic cat and clouded leopard). The intra- and interspecific variations of these four mapped species ("index species") have been measured. The mtDNA of the domestic cat has been molecularly cloned in the plasmid, pBR322. The mtDNA clones will be used as molecular probes of mtDNA from each Felidae species (35 of 37) using "Southern" analysis of high molecular weight DNA from cultured cell lines. The comparative restriction maps of the mtDNA from the various species will be used to construct a molecular phylogeny of the Felidae. This phylogeny will be compared to phylogenetic topologies of the Felidae derived by other molecular and morphometric measurements.