During asymmetric cell division, the membrane-associated Numb protein localizes to a crescent in the mitotic progenitor and is segregated predominantly to one of the two daughter cells. We have identified a putative serine/threonine kinase, Numb-associated kinase (Nak), which interacts physically with the phosphotyrosine binding (PTB) domain of Numb. The PTB domains of Shc and insulin receptor substrate bind to an NPXY motif which is not present in the region of Nak that interacts with Numb PTB domain. We found that the Numb PTB domain but not the Shc PTB domain interacts with Nak through a peptide of 11 amino acids, implicating a novel and specific protein-protein interaction. Overexpression of Nak in the sensory organs causes both daughters of a normally asymmetric cell division to adopt the same cell fate, a transformation similar to the loss of numb function phenotype and opposite the cell fate transformation caused by overexpression of Numb. The frequency of cell fate transformation is sensitive to the numb gene dosage, as expected from the physical interaction between Nak and Numb. These findings indicate that Nak may play a role in cell fate determination during asymmetric cell divisions.