The proper folding of tubulins and their incorporation into microtubules consist of a series of reactions, in which evolutionarily conserved proteins, cofactors A to E, play a vital role. We have cloned a fission yeast gene (alp41(+)) which encodes a highly conserved small GTP-binding protein homologous to budding yeast CIN4 and human ARF-like Arl2. alp41(+) is essential, disruption of which results in microtubule dysfunction and growth polarity defects. Genetic analysis indicates that Alp41 plays a crucial role in the cofactor-dependent pathway, in which it functions upstream of the cofactor D homologue Alp1(D) and possibly in concert with Alp21(E).