Several genes that play roles in vacuolar biogenesis and targeting of proteins to vacuoles have been characterized in Saccharomyces cerevisiae. Although the vacuole is one of the most prominent compartments, little is known about molecular mechanism of vacuolar biogenesis in filamentous fungi. Vam4/Ypt7p, a small GTPase of the Rab/Ypt family in S. cerevisiae, plays a vital role in homotypic vacuole fusion. We describe the isolation of the avaA gene from Aspergillus nidulans as a homologue of the VAM4/YPT7. Predicted 205 amino acids protein encoded by the avaA showed 67 and 72% identity with yeast Vam4/Ypt7p and human Rab7, respectively. The avaA disruptants exhibited highly fragmented vacuoles. We introduced mutations into the avaA gene, which alter nucleotide-binding characteristics of the gene products. Replacement of the avaA gene to the GDP-bound form mutant resulted in fragmentation of vacuoles. Overexpression of the GTP-bound form of avaA severely inhibited the hyphal growth and caused abnormal swelling of vacuoles. These results suggest that A. nidulans AvaA functions in the vacuolar biogenesis.