The haplophase of the pine blister rust Cronartium flaccidum was used to grow mycelial clones axenically from single genotypes. Single telia were suspended over the media in order to obtain a direct cast of uninucleate basidiospores, providing nurse cultures from a mass basidiospore inoculum. Nurse culture and medium composition were crucial factors in successful germination and establishment of single-spore colonies. Significant differences in percentage germination between treatments and controls suggested that an extracellular matrix secreted by high density seeded basidiospores could be involved in the germination of single propagules. Proteins present in this matrix could exert a surface contact stimulus triggering germination of newly seeded basidiospores. Pre-production of extracellular enzymes in the medium could also initiate early substrate catabolism and thus create a favourable environment to support subsequent colony establishment. Growth was good on various media, and excellent on HG1Y+BSA medium. Primary mother colonies were divided into several smaller ones, re-grown axenically, then macerated. Aliquots of the resulting mycelial fragments were dispensed with a micropipette into fresh agar media. Clones derived from each pure culture were in general morphologically similar in texture, margin, compactness, and surface topography. These genetically pure lines grew at a faster rate and rapidly produced a considerable amount of mycelium. The significance of culturing rust fungi from single genotypes is discussed in relation to potential applications and new directions of research.