Attempts were conducted to establish in vitro vesicular-arbuscular mycorrhizal (VAM) associations under gnotobiotic conditions. Alnus glutinosa (L.) Gaertn. served as the host in the form of whole plants, root organs, and undifferentiated callus. Spores of Gigaspora rosea Nicolson and Schenck or Glomus etunicatum Becker and Gerdemann were utilized as VAM fungus (VAMF) inoculum. Limited VAM associations were attained within certain in vitro culture systems that contained intact seedlings or root organs. In addition, whole seedling cultures exhibited occasional sporulation by Gl. etunicatum;The influences of chloramphenicol (150, 75, and 35 ppm), gentamicin sulfate (100 and 50 ppm), streptomycin sulfate (50 and 25 ppm), and penicillin G potassium (50,000 and 25,000 units/L) upon spore germination and hyphal growth by the two VAMF species also were evaluated in 1% water agar. Germination of Gl. etunicatum chlamydospores was inhibited by gentamicin, and hyphal growth was sensitive to higher concentrations of chloramphenicol and gentamicin. Higher levels of chloramphenicol also reduced germination and hyphal growth of Gi. rosea. Effects of other antibiotics were dependent upon their concentration and VAMF species. No significant correlation was found between spore diameter and hyphal growth for either VAMF species. Hyphal growth by Gi. rosea was, however, highly correlated with the number of extra-matrical vesicle clusters that were produced upon each hypha. When extra-matrical vesicle clusters became separated by a septum from the parental hypha, they occasionally generated a new radial mass of thin hyphae. Chlamydospores of Gl. etunicatum occasionally germinated directly through the spore wall, and hyphae from separate spores in close proximity frequently anastomosed;Light and scanning electron microscopy were utilized to observe various anatomical aspects in the development of Gi. rosea and Gl. etunicatum. These VA mycobionts were examined in an independent state and in association with roots of A. glutinosa.