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 KIGal80p, can also inhibit the heterologous Gal4p variants; however, heterologous Gal3p/Gal1p only regulate ScGal80p but not KIGal80p. To compare KIGal80p and ScGal80p, point mutations known to affect ScGal80p function were introduced at corresponding positions in KIGal80p, and Gal4p regulation in vivo and KIGal80p-binding to Gst-Gal1p and Gst-Gal4-AD in vitro were analysed. The in vitro binding properties of the KIGal80p mutants were similar to those of ScGal80p, but two out of four mutants differed in Gal4p regulation. E. g. KIGAL80s-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 KIGal80p are shown to be subject to carbon source regulated phosphorylation whereas no evidence for ScGal80p phosphorylation exists. (Hyper-)phosphorylation of KIGal80p is strongly reduced in galactose-containing medium. This reduction requires KIGal1p but no interaction with KIGal4p. The inhibition deficient KIGal80s-0p (G302R) variant is under-phosphorylated. We thus propose that phosphorylation of Gal80p in Kluyveromyces lactis contributes to the regulation of Gal4p mediated transcription.