Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.