A kinase activity that phosphorylated myelin basic protein in vitro was detected in RalA and RalB immunoprecipitates from human platelets. Protein-protein interaction studies using recombinant GST-RalA, GST-RalB and GST-cH-Ras confirmed that the kinase specifically associates with the Ral GTPase. The Ral Interacting Protein 1 (RIP1), a GTPase Activating Protein (GAP) for Cdc42 and Rac1, was found to be the preferred substrate for the Ral Interacting Kinase (RIK). Phosphoamino acid analysis demonstrated that RIK phosphorylated serine residue in RIP1. The Ral-RIK interaction was not dependent on the guanine nucleotide status of Ral. RIK was detected in a variety of rat tissues with testis containing the highest and skeletal muscle the lowest activity. In-gel kinase renaturation assay using RIP1 as the substrate demonstrated that the kinase activity was associated with polypeptides of molecular mass of approximately 36-40 kDa and was detected in most rat tissues with a prominent 38 kDa band in testis and a 40 kDa band in brain. Human platelets contained a single band of approximately 36 kDa. RIK was distinct from MAPKs, CDKs, cyclic AMP dependent protein kinase and Ca2+/calmodulin dependent kinases. To demonstrate in vivo interaction, the endogenous Ral-RIK complex was isolated using a calmodulin affinity column. The Ral-RIK complex co-eluted from this column upon washing with a 13 residue peptide that encompasses the calmodulin-binding domain in RalA. The data suggest that RIK is a serine specific kinase that phosphorylates RIP1 and is constitutively associated with Ral. The current study provides additional support for a link between Ral and the Cdc42/Rac1 signalling pathways in the cell.