The yeast Gal80 protein inhibits the transcription activation function of Gal4p by physically interacting with the activation domain (Gal4-AD). Gal80p interaction with Gal1p or Gal3p is required to relieve Gal4p inhibition in response to galactose. Gal80p orthologs of Saccharomyces cerevisiae and Kluyveromyces lactis, ScGal80p and KlGal80p, can also inhibit the heterologous Gal4p variants; however, heterologous Gal3p/Gal1p only regulate ScGal80p but not KlGal80p. To compare KlGal80p and ScGal80p, point mutations known to affect ScGal80p function were introduced at corresponding positions in KlGal80p, and Gal4p regulation in vivo and KlGal80p-binding to Gst-Gal1p and Gst-Gal4-AD in vitro were analysed. The in vitro binding properties of the KlGal80p mutants were similar to those of ScGal80p, but two out of four mutants differed in Gal4p regulation. E. g. KlGAL80s-0 (G302R) but not ScGAL80s-0 (G301R) alleviates Gal4p inhibition. Possibly, this difference is related to a role of phosphorylation in the regulation of Gal80p function in K. lactis. Wild-type and mutant forms of KlGal80p are shown to be subject to carbon source regulated phosphorylation whereas no evidence for ScGal80p phosphorylation exists. (Hyper-)phosphorylation of KlGal80p is strongly reduced in galactose-containing medium. This reduction requires KlGal1p but no interaction with KlGal4p. The inhibition deficient KlGal80s-0p (G302R) variant is under-phosphorylated. We thus propose that phosphorylation of Gal80p in Kluyveromyces lactis contributes to the regulation of Gal4p mediated transcription.