DNA fragments containing genetic information for five secretion-related small GTPases of Aspergillus niger (srgA-E) were isolated and identified as members of different Rab/Ypt subfamilies. This isolation and the search for similar sequences in fungal genomic and EST databases showed that, in contrast to Saccharomyces cerevisiae, filamentous fungi also possess homologues of mammalian Rab2 GTPases. Multiple transcripts with unusually long 5′ and 3′ untranslated regions were found for all srg genes. Their level of expression was independent of the type of carbon source used for growth. Although the transcripts of srgA and srgB were abundant to the same extent throughout the cultivation, that of the other genes peaked during the early growth phase and then declined. Two genes, srgA and srgB, were characterized further. The protein encoded by srgA exhibited relatively low identity (58%) to its closest S. cerevisiae homologue SEC4, whereas the protein encoded by srgB showed 73% identity with S. cerevisiae YPT1. In contrast to other SEC4 homologues, srgA was unable to complement an S. cerevisiae sec4 mutant, and its disruption was not lethal in A. niger. SrgA mutants displayed a twofold increase in their hyphal diameter, unusual apical branching and strongly reduced protein secretion during growth on glucose. Molecular Sequence Numbers: A44334, AJ224685, AJ278657, AJ278658, AJ278659, AJ278660, AJ278661, AJ278662, AJ404733, AJ404734, P07560, S32965, X52099, X52100, X52469, X52475, X59598, X72833, X72834, X76173, X76174, X76175, Z22220, Z98598; Chemicals/CAS: Fungal Proteins; Glucose, 50-99-7; maltodextrin, 9050-36-6; Polysaccharides; rab GTP-Binding Proteins, EC 3.6.1.-; Saccharomyces cerevisiae Proteins; SEC4 protein, S cerevisiae, EC 3.6.1.-.