Knight, J. D. 2015. Delayed inoculation of alfalfa with Sinorhizobium meliloti and Penicillium bilaiae. Can. J. Plant Sci. 95: 205-211. The persistence of perennial forage legume crops relies on the establishment of an effective symbiotic relationship with the appropriate Rhizobium species and strain. Situations can arise where a forage legume crop fails to symbiotically fix N₂. This study investigates if inoculation of alfalfa with a commercial Sinorhizobium meliloti inoculant 1 yr after seeding can induce biological N₂ fixation at levels similar to those achieved when the inoculant is applied at seeding. Alfalfa (Medicago meliloti cv. Algonkwin) was grown at two sites in Saskatchewan and inoculated with S. meliloti or S. meliloti plus the P-solubilizing fungus Penicillium bilaiae. The inoculants were applied at seeding or applied 1 yr after seeding. Biological N₂ fixation was measured in the fall of the delayed inoculation year as percentage of N derived from atmosphere (%Ndfa) using the ¹⁵N isotope dilution technique. Inoculation with S. meliloti increased %Ndfa at both sites relative to uninoculated and fertilized controls but had no effect on total N content or yield. Inoculating alfalfa the year after seeding increased %Ndfa relative to the controls at both sites, and at one of the sites %Ndfa in the delayed treatments was at the same levels measured in the year of seeding treatments.