Despite several previous studies focusing on osmostress response in the extremely halotolerant black yeast Hortaea werneckii, the key role of the MAP kinase HwHog1 remains unsolved. HwHog1 activation is regulated by the HOG signaling pathway, through which the signal is transferred from the membrane osmosensors to the MAP kinase cascade inside the cell. This triggers HwHog1 phosphorylation, which leads to its activation and regulation of expression of target genes and activity of target proteins. To resolve this question, we wanted to employ a chemical-genetic approach using a novel inhibitor of the native Hog1 kinase, a Saccharomyces cerevisiae homologue of the HwHog1 kinase. To this end, we constructed a yeast plasmid with inserted intronless HwHOG1 gene and transformed it into the S. cerevisiae strain with deleted HOG1 gene. Western blotting confirmed the presence of the HwHog1 in the transformed cells. The described system enabled us to study the effect of the inhibitor in the same genetic background as was used for the Hog1 inhibitor testing. Functional complementation assays and western blotting using antibodies against phospho(p38) showed, that HwHog1 was able to complement the MAP kinase Hog1 function in S. cerevisiae Δhog mutant and rescue the osmosensitive phenotype. Evidently, HwHog1 is able to accept the signal from the MAPK kinase Pbs2 and translocate to the nucleus where it regulates the expression of several genes, enabling S. cerevisiae to adapt to the increased osmolarity in the environment. Moreover, the inhibitor plate tests showed that PD447, which is an effective in vivo inhibitor of the Hog1 MAP kinase, efficiently inhibits the HwHog1 kinase when expressed in S. cerevisiae. Altogether we showed the suitability of the use of inhibitor PD447 in the chemical-genetic approach for studying the role of HOG response pathway in H. werneckii. In the future we want to examine the effect of the inhibitor on the HwHog1 kinase activity in H. werneckii and hopefully demonstrate the essential role of the HOG pathway in osmostress response in the black yeast H. werneckii.