The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library. A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase II, and a P. guilliermondii rib1 mutant was isolated and sequenced. An open reading frame with the potential to encode a protein of 344 amino acids with a predicted molecular mass of 38,711 Da was detected. The P. guilliermondii enzyme shows a high degree of homology to GTP-cyclohydrolases type II from E. coli and Baccillus subtilis and to GTP-cyclohydrolase from Saccharomyces cerevisiae. Functional GTP-cyclohydrolase from P. guilliermondii may consist of four identical subunits.