Pheromone signalling during mating is essential for pathogenicity of Ustilago maydis. The activity of the key transcription factor Prf1 is controlled at the transcriptional level and post-translationally by mitogen-activated protein kinase (MAPK) and protein kinase A (PKA) phosphorylation. However, the precise contribution of these regulatory mechanisms to the transcriptional output is unknown. Here, we genetically dissected the three levels of Prf1 regulation. We performed transcriptional profiling of respective mutants to identify and classify targets. This approach revealed that transcriptional regulation of prf1 had only minor influence on target gene expression stressing the importance of post-translational control. PKA regulation of Prf1 was sufficient to control expression of nine pheromone-responsive genes including the major transcription factor regulating pathogenicity. MAPK regulation was necessary for the pheromone response of a set of 57 genes. In 35 cases, pheromone responsiveness was completely lost, while in the remaining 22 cases regulation was alleviated. This indicated a novel level of complexity in MAPK signalling suggesting that target genes respond differentially to MAPK phosphorylation of the respective transcription factors.