Rhodococcus equi is a facultative pathogen which infects the lower respiratory tract of foals and immune-supressed humans. R. equi infection usually occurs through aerosol inhalation and leads to pyogranulomatous pneumonia. Two component regulatory systems are critical for niche adaption and therefore for pathogen survival in the host. Environmental signals are typically mediated by a sensor kinase, which phosphorylates a partner response regulator. In turn, the response regulator regulates transcription of target genes. Genes encoding sensor kinase and response regulator are most times in the same operon. VirS is an orphan response regulator of R. equi. It is essential for regulation of genes encoding the virulence associated proteins (vaps) and for bacterial replication inside macrophages. The genome of R. equi encodes 24 sensor kinases, of which all but one have their cognate response regulator. Given the specificity of the interaction between sensor kinase and response regulator, yeast two-hybrid screening was employed to identify the partner senor kinase of VirS. We found that VirS interacts with at least two sensor kinases. We then employed gene deletion analysis to determine whether the identified sensor kinases are required for the regulation of vapA and the survival of R. equi in macrophages. Neither single nor multiple deletion mutants showed any effect on intracellular proliferation. Therefore, it is highly likely that there is redundancy of partner sensor kinases of VirS and at least one more sensor kinase partner need to be identified.