Acremonium cellulolyticus CF-2612 is a cellulase hyper-producing mutant that originated from A. cellulolyticus Y-94. In this study, we isolated a uracil auxotroph (strain CFP3) derived from CF-2612, and cloned a wild-type pyrF gene encoding orotate phosphoribosyl transferase (OPRTase) from Y-94. OPRTase activity was not detected in strain CFP3, which had one nucleotide substitution in its pyrF gene. The wild-type pyrF gene restored the defective growth of CFP3 on uracil-free medium, and PCR and Southern analyses revealed that wild-type pyrF was integrated into the genome. These results indicate that our transformation system for A. cellulolyticus with the pyrFgene as a selection marker was successful.