Abstract 1. 1. Cell-free preparations of Saccharomyces cerevisiae, were shown to catalyze the reduction of 2-methyl-4-amino-5-formylpyrimidine to the corresponding 5-hydroxymethyl derivative, a known intermediate in thiamine biosynthesis. Either NADH or NADPH served as a cofactor. The pH optimum of the system was 6.5–7.0 and the apparent K m for the formylpyrimidine substrate was 3.3·10 −4 M. 2. 2. Details of a microbiological assay for the pyrimidine moiety of thiamine, using Escherichia coli Strain M70-17, are described.