AbstractNKR‐P1 is a type II transmembrane protein which acts as an activation receptor on natural killer (NK) cells. The cytoplasmic domains of the CD4, CD8 and 4‐1BB receptors contain the sequence Cys‐X‐Cys‐Pro which is directly involved in coupling to another pair of cysteines in the N‐terminal domain of the src family tyrosine kinase p56lck. The cytoplasmic domain of NKR‐P1 in rodents also contains the Cys‐X‐Cys‐Pro sequence, but the capacity of the receptor to bind p56lckis presently unknown. We tested for direct coupling between these proteins using both protein biochemistry and the yeast two‐hybrid technique. Immunoprecipitation studies showed that p56lckcan be co‐immunoprecipitated with NKR‐P1 from a rat NK tumor cell line. In addition, the cytoplasmic domain of NKR‐P1 interacted with the N‐terminal domain of p56lckin yeast as assessed by reporter gene activation. Integrity of the cysteine pairs in both proteins was critical in mediating the interaction. The experiments suggest that the association of p56lckwith NKR‐P1 is somewhat weaker than the p56lckassociation with CD8α, but of much lower avidity than between CD4 and p56lck. This could reflect a higher activation threshold for the NKR‐P1 and CD8 receptors, which are involved in cytolytic responses, compared to CD4 which is