Here we investigated the involvement of HS1, the hematopoietic cell-specific homolog of cortactin, in the actin-based functions of natural killer cells. Involvement of HS1 in T cell regulation has been established, as HS1 is required for the formation of immune synapses. 'Knockdown' of HS1 in natural killer cells resulted in defective lysis of target cells, cell adhesion, chemotaxis and actin assembly at the lytic synapse. Phosphorylation of the tyrosine residue at position 397 (Tyr397) was required for adhesion to the integrin ligand ICAM-1 and for cytolysis, whereas phosphorylation of Tyr378 was required for chemotaxis. Phosphorylation of Tyr397 was also required for integrin signaling and recruitment of integrins, adaptors and actin to the lytic synapse. Thus, HS1 is essential for signaling and actin assembly in natural killer cells, and the functions of the two phosphorylated tyrosine residues are distinct and separable.