The nature of products formed from GTP in the presence of NADPH under effects of dialyzed extracts of the prototrophic strain of Pichia guilliermondii ATCC 9058 and mutants rib2 and rib3 with blocked second and third steps of flavinogenesis, respectively, was studied. The unstable pyrimidine intermediates were identified on the basis of the nature of pteridines formed upon condensation of riboflavin precursors with diacetyl. It was shown that under the action of extracts of the mutants rib3 GTP was predominantly converted into 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine phosphate. In similar studies on the extracts of the prototrophic strain, 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine phosphate, 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine and trace amounts of 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine were detected. No ribitylated intermediates of riboflavin were formed in the extracts of rib2 mutants. The results obtained are indicative of the participation of phosphorylated pyrimidine precursors in the second and third steps of flavinogenesis in yeasts. 6,7-Dimethyl-8-ribityllumazine phosphate is not the substrate for P. guilliermondii riboflavin synthase.