Actinorhizal nodules of Alnus incana ssp. rugosa collected from plants growing within 30 km of each other in Jeffersonville and Stowe, VT, U.S.A., yielded two morphologically distinct strains of Frankia. A comparison of the cell chemistry of the strains showed that each had the same type of cell wall and whole-cell sugar and phospholipid composition; however, AirI2 lacked the unknown amino acid found in the cells of all frankiae previously examined. Their cellular fatty acid composition showed only minor qualitative differences. Physiologically, the strains differed in their relation to oxygen, in their uptake and utilization of carbohydrates, organic acids, and lipids, and in protease production. AirI1 induced effective nodules in the host plant; those of AirI2 were ineffective. AirI2 was unrelated serologically to all frankiae previously isolated from the genus Alnus. AirI1 was serologically typical.