Spt3 of Saccharomyces cerevisiae is a factor required for normal transcription from particular RNA polymerase II-dependent promoters. As a step towards analysing Spt3 structure-function relationships, we have identified and studied Spt3 homologues from three other yeasts: Kluyveromyces lactis, Clavispora opuntiae and Schizosaccharomyces pombe. Alignment of their predicted amino acid sequences shows an overall identity of 30% between all four homologues and suggests that three conserved domains are present in Spt3. When tested for function in S. cerevisiae, K. lactis SPT3 was shown to fully complement and S. pombe SPT3 to partially complement an spt3 delta mutation. These data demonstrate that Spt3 is functionally conserved among distantly related yeasts.