Abstract:It is very important to select carbon and nitrogen sources for isolating the endophytes from nodules of actinorhizal plants which are nonlegumen. Tween homologues, including Span, may be able to become a good C-source in the isolation and cultivation of Frankia species. Some strains showed better behaviour of growth on pyruvic acid than on propionic acid currently used. It was noticed that vitality of the inoculation should be kept in the procedures of isolation. It was also found that simple and fast techniques of inoculation were more successful than those with complex procedures. The range of tested concentrations of ABA and phenolic compounds didn‘t obviously inhibit Frankia strains from their growth and isolation. Actinorhizal endophytes could be isolated from the nodules in late spring, summer, autumn, and unisolated in the winter. Fresh nodules should be selected for isolationtobe successful. pH value was not a critical fact in the procedures of isolation.