The transcriptional regulatory protein Leu3 of Saccharomyces cerevisiae was enriched approximately 70-fold above wild type level in yeast cells carrying a pGAL1-LEU3 expression vector. Sustained overproduction of Leu3 following induction by galactose required elevated intracellular levels of alpha-isopropylmalate, a leucine pathway intermediate known to act as transcriptional co-activator. Starting with galactose-induced cells, the Leu3 protein was purified about 3,500-fold (i.e. 245,000-fold over wild type level) by a procedure that included treatment of the cell-free extract with polyethylenimine, fractionation with ammonium sulfate, heat treatment, and DNA affinity chromatography. Highly purified preparations still showed two protein bands when subjected to polyacrylamide electrophoresis under denaturing conditions. Their apparent molecular masses were about 104,000 and 110,000 kDa. The smaller of these values was very close to the maximum molecular weight obtained previously for Leu3 protein translated in vitro in a rabbit reticulocyte lysate. (The molecular weight deduced from the open reading frame of the LEU3 gene is 100,162.) Both protein bands reacted with antibodies raised against different portions of the Leu3 molecule and were, therefore, likely to represent two forms of Leu3. Treatment with calf intestinal phosphatase quantitatively converted the slower moving band into the faster moving one. Conversion was prevented by inorganic phosphate, a phosphatase inhibitor. These experiments showed that the two bands very likely correspond to phosphorylated and nonphosphorylated forms of Leu3. Phosphorylation did not appear to affect the DNA binding function of Leu3, but (indirect) effects on the activation function or effects on the modulation by alpha-isopropylmalate have not been ruled out. Electrophoretic mobility shift assays were used to estimate the apparent dissociation constants of the two specific Leu3-DNA complexes routinely seen in these assays. The values obtained were 1.1 and 2.6 nM. Finally, using size exclusion chromatography, native Leu3 protein was shown to have dimeric structure, irrespective of the state of phosphorylation.