Alfalfa (Medicago sativa L.) plants were produced from callus tissue cultured on a variety of semisolid media. Plants could be obtained from callus placed on a basal medium, but addition of naphthalene acetic acid, tyrosine, or a cytokinin either alone or in certain combinations increased efficiency. The most efficient production was accomplished on a medium composed of Blaydes' inorganic and vitamin constituents with 100 mg/liter inositol and 2.0 g/liter yeast extract. Ability of fresh callus to differentiate into plantlets depended on the hormone levels of the initial callus‐inducing medium, as well as on the genotype of the donor plant. Daylength appeared not to be critical for shoot differentiation. Plants differentiated out of callus derived from immature anthers, internode sections, seedling hypocotyls, and immature ovaries. Callus was obtained from anthers which were in premeiotic to nearly mature stages. Histological studies indicated that in most cases anther callus arose from somatic cells in interlocular connective tissue. Most of the 200 plants recovered from callus were tetraploid, as was the donor plant, although at least nine octaploids and several off‐type plants, including albinos, were obtained.