We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.