To isolate full length cDNA clones encoding human GTP cyclohydrolase I, the first and rate-limiting enzyme in tetrahydrobiopterin biosynthesis, a cDNA library generated from human liver was screened by plaque hybridization. Analysis of the clones, hybridized with rat cDNA fragment, by restriction mapping and partial sequencing showed the existence of three kinds of cDNAs. All three cDNAs were identical in their central and 5' regions. They were, however, found to diverge at 3' ends. Furthermore, the three species of mRNAs corresponding to the three cDNAs were detected in human liver by reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results indicate that, in humans, GTP cyclohydrolase I molecules are encoded by at least three distinct mRNAs.