Carotenoids are a naturally occurring and widely distributed group of pigments that provide protection against photooxidation and inactivate free radicals due to their highly conjugated double-bond systems. Phytoene dehydrogenation is the first rate-limiting step in the carotenoid biosynthetic pathway. Phytoene dehydrogenase is the key enzyme in the transformation of carotenoid from colorless to colored; therefore it is the first target of gene manipulation. The present study describes the identification and functional characterization of a carontenoid synthesis gene from Rhodosporidium diobovatum, designated as crtI, which catalyzes the dehydrogenation of phytoene. We obtained a full-length cDNA clone of crtI, encoding phytoene dehydrogenase (EC Number: 1.3.99.31), from R. diobovatum ATCC 2527 by rapid amplification of cDNA ends. Complementation mapping of the crtI gene in Escherichia coli allowed us to localize the regions responsible for phytoene dehydrogenase function within the protein. Enzyme activity of the expressed protein in E. coli was verified using high performance liquid chromatography analysis. We were able to determine the nucleotide sequence of crtI from R. diobovatum. The publicly available sequence will be useful in future studies on phytoene dehydrogenase.