The Arabidopsis thaliana Histidine Kinase 1 (AHK1) is part of the multistep phosphorelay system and stands at the beginning of a signaling cascade. Phylogenetic analysis showed that AHK1 and also its ecto-domain (ED) is highly conserved in dicot plants, like A. thaliana and Lotus japonicus. A homology-based structural model revealed that the AHK1ED might comprise a Per-Arnt-Sim (PAS) domain. A PAS-domain is known for binding low molecular weight ligands. It is similar to PAS-like CACHE domains that other AHK’s carry, and which is associated with the phytohormone cytokinin. In this study we tried to identify the ligand of AHK1. For identification of the AHK1 ligand, the ED of AHK1 is expressed in E. coli, purified and used for ligand-fishing via LC-MS. In addition, we used a microscopic approach in which we expressed transiently full-length AHK1 in plant leaves. The plant leaves were than treated with our candidates or with the inhibitors of our candidates. As shown by a quantitative phosphoproteomics approach, the activation of AHK1 led to the rapid phosphorylation of many proteins. The identified proteins were involved in, for instance, stress and light signaling. Therefore, we pursued to characterize the main pathway of AHK1. We executed phenotypic analyses using Arabidopsis seedlings carrying different ahk1 mutant alleles based on the findings. Hereby we applied different environmental cues e.g. irradiation with light of different intensity and wavelengths, application of different stress conditions, which are linked to proteins differentially phosphorylated by AHK1. In conclusion, our analysis will help to understand the molecular process underlying the activation of AHK1. Furthermore, we could describe two homologs of AHK1 in L. japonicus, which we called LHK4-1 and LHK4-2. In their alleles we could find similarities and differences to ahk1. Our phenotypic analysis in Arabidopsis could further elucidate the signaling network in which AHK1 is embedded. Our data indicate that AHK1 is involved in lipid signaling but we were not able to identify a lipid as AHK1’s signal.