Precursor incorporation experiments have ruled out the involvement of N'-formylkynurenine in the biosynthesis of the uracil acrylic acid moiety of sparsomycin. An enzyme has been partially purified from the sparsomycin-producer that catalyzes the synthesis of the uracil acrylic acid from a pyrimidine acrylic acid previously shown to be a sparsomycin precursor. The enzymatic synthesis of the uracil acrylic acid requires NAD+, a result which indicates that the mechanism of the uracil acrylic acid synthase is similar to that of inosine-5'-monophosphate dehydrogenase. Analog studies demonstrate that the enzyme lacks rigid substrate specificity.