In the yeast Saccharomyces cerevisiae regulation of the galactose/melibiose regulon rests on a dosage-dependent functional interplay between the positive regulator of transcription, the GAL4 protein, and the negative regulator, GAL80 protein. We have used this interplay to select in vitro generated fusions between the yeast ADH1 promoter and GAL4 coding sequence that overproduce GAL4 protein, allowing the identification of GAL4 protein in crude extracts from yeast. One type of these constructions produces a GAL4 protein that lacks its normal NH2 terminus. This protein is unable to complement a gal4 lesion but still retains a domain that functionally antagonizes the negative regulatory protein. One defect in this truncated protein is its inability to be concentrated in the nucleus. However, the nuclear localization defect is complemented by full-length GAL4 protein. The truncated protein also appears to effect changes in the relative transcriptional levels of the structural genes. These observations imply that GAL4 protein consists of several domains, including ones for nuclear localization, interaction with the negative regulatory protein, and, possibly, separable transcriptional activation domains for the structural genes.