The filamentous fungus Aspergillus nidulans has two genes encoding α-tubulin, tubA and tubB, which are differentially required at distinct stages during the life cycle. The tubA gene is required during vegetative growth for mitosis and nuclear migration (B. R. Oakley, C. E. Oakley, and J. E. Rinehart, Mol. Gen. Genet. 208:135-144, 1987; P. Doshi, C. A. Bossie, J. H. Doonan, G. S. May, and N. R. Morris, Mol. Gen. Genet. 225:129-141, 1991). The tubB gene is not required for any detectable aspect of vegetative growth or asexual reproduction but is essential during sexual development prior to the first meiotic division (K. E. Kirk and N. R. Morris, Genes Dev. 5:2014-2023,1991). In this study, we determined whether the role of each α-tubulin gene is to provide a specific isotype necessary for a particular microtubule function or whether either α-tubulin isotype, if present in sufficient quantities, can participate effectively in all types of microtubule. Strains carrying a deletion allele of tubB (tubBΔ) produce no ascospores from a cross. When one copy of a plasmid containing the region upstream of the tubB gene fused to the tubA coding region was integrated into a tubBΔ strain, ascosporogenesis proceeded beyond the tubBΔ block and resulted in the formation of sexual spores. However, irregular numbers of spores formed in some asci during development, and the ascospores had greatly diminished viability and aberrant morphologies. These defects were nearly corrected when two additional copies of the tubA coding region were integrated into the tubBΔ strain. These results indicate that the tubA α-tubulin isotype can form functional microtubules during sexual development in the absence of tubB protein. In a reciprocal set of experiments, we examined whether upregulation of tubB can complement the tubA4 mutation, which causes supersensitivity to benomyl during vegetative growth. When tubA4 strains integrated a plasmid containing an alcohol-inducible promoter joined to the tubB coding region and subsequently overexpressed the tubB isotype, the benomyl supersensitivity normally caused by the tubA4 allele was relieved. These results indicate that when enough tubB α-tubulin is supplied, strains lacking functional tubA isotype can still form microtubules which effectively carry out mitosis and nuclear migration.