L-ascorbic acid, vitamin C for those species dependent on an exogenous source, is a physiologically essential molecule for all multicellular animals. Nevertheless, some species of birds and mammals lack and apparently have lost the capacity to synthesize it. Some vertebrate species synthesize it in liver, some in kidney, and some in both. It has been the goal of our research for several years to understand the evolution of the ascorbic acid pathway in vertebrates, as a model for the study of (1) regressive (=streamlining) evolution, (2) tissue locus shifts in biochemical pathways, and (3) the operation of selection on the ability of animals to regulate the production of an essential molecule. We here present data on the activity and tissue locus of L-gulonolactone oxidase (GLO) (EC1.1.3.8) in the Monotremata and Marsupialia and integrate these data into an hypothesis of the evolution of ascorbate biosynthesis in vertebrates. GLO catalyzes the final step, conversion of L-gulonolactone to L-ascorbic acid, in the pathway by which the latter is synthesized from D-glucose (Dagley and Nicholson, 1970; Chatterjee, 1973a). Although few species of most higher taxa have been examined, invertebrates (Gupta et al., 1972; Chatterjee, 1973b), teleost fish (Chatterjee, 1973a; Andrews and Murai, 1975), some birds (Chaudhuri and Chatterjee, 1969), and a few mammals (Chatterjee, 1973a), appear to lack GLO, and thus are unable to synthesize ascorbate. Lungfish (D. E. Dykhuizen, pers. comm.), amphibians, reptiles, and most birds are reported to have GLO in the kidney whereas eutherian mammals and some birds have it in the liver (Chatterjee, 1973a). We recently demonstrated (Birney et al., 1979) GLO activity in kidney of monotremes and in both liver and kidney in bandicoots and in some macropods. Prior to that report, the Virginia opossum (Didelphis virginiana) was the only noneutherian mammal studied, and it was believed to be capable of synthesizing ascorbate in liver on the basis of dehydrogenation of L-gulonolactone as determined histologically (Nakajima et al., 1969). The basic evolutionary relationship and times of divergence for the three major groups of mammals, Prototheria, Metatheria, and Eutheria, are reasonably well understood (Hopson and Crompton, 1969; Crompton and Jenkins, 1973; Lillegraven, 1974; Clemens, 1977). Furthermore, Ride (1964), Kirsch (1977a, 1977b), and Kirsch and Calaby (1977) have greatly enhanced our knowledge of the evolutionary relationships within the Marsupialia. Thus we feel that the relationships of the major groups of mammals discussed herein are adequately known so that we can superimpose the pattern of GLO activity on this phylogeny and initiate an understanding of the evolution of the biochemical system involved by proposing and evaluating some preliminary hypotheses.