Axenic cultures of the aspen rust Melampsora larici-tremulae were established for the first time by seeding aeciospores artificially produced on larch onto three chemically defined media. Cultures were maintained for 9 months and evaluated for their appearance, growth rates and sporulation ability. Two types of colonies were observed. One, white and fluffy tending to felty, did not sporulate in culture. The other, white-orange with a more compact aerial mycelium, sporulated profusely producing various spore like bodies and other unusual structures. An initial lag phase, with reduced growth, characterised primary rust cultures in the first 4–5 weeks. After about one month, cultures seemed adapted to utilising exogenous nutrients for sustained growth and their development rate increased substantially. As a result of selection under unusual growing conditions, fast-growing genetic variants were also observed in the nutrient agar. Growth on all the three test media, containing different organic and inorganic constituents, suggested that nutritional requirements were non-specific. The importance of axenic cultures in the study of the nutritional requirements of aspen rust and the potential of these investigations to elucidate some physiological and genetic aspects of the host-parasite relationship are discussed.