SUMMARY. An improved assay of vitamin B12 is described. The z strain of Euglena gracilis, which grows more vigorously than the bacillaris strain previously used, is recommended. The pattern of B12 specificity of the two strains appears to be the same. A new medium containing sucrose, aspartic acid, glutamic acid, and glycine at pH 3.6 is well buffered and allows luxuriant growth. Vigorous utilization of sugar appears to depend on readily available nitrogen and a Krebs‐cycle component; these requirements are well met by aspartic acid (or asparagine) and glutamic acid. The proposed procedure is especially suitable for the measurement of B12 in blood serum because rise in pH and precipitation of serum proteins during incubation are minimized. Like bacillaris, the z strain allows the distinction between “combined” and “uncombined” B12 in serum. Serum may be an appropriate test material to tell whether such phagotrophs as Peranema can better utilize bound forms of vitamins than can the related osmotrophs. Patterns of B12 requirements and occurrence are discussed as phylogenetic markers.