The chloroplast stromal proteins from spinach condense the two heterocyclic moieties of vitamin B1--4-methyl-5-(beta-hydroxyethyl)thiazole and 2-methyl-4-amino-5-hydroxymethylpyrimidine--in the presence of MgATP. We have taken advantage of this observation to study thiazole synthesis. We present data indicating that pyruvate, glyceraldehyde 3-phosphate, tyrosine, cysteine, and MgATP are required for this synthesis and that pyruvate and glyceraldehyde 3-phosphate can be replaced by 1-deoxy-D-threo-2-pentulose. The thiazole synthesis occurs at a sustained, low rate of 140 pmol per mg of protein per hr.