Natural killer (NK) cells can express high levels of CD44, and signaling through CD44 has been shown to enhance NK cell cytotoxic activity. However, little is known about the factors that regulate CD44-mediated activation of NK cells. The studies reported here reveal that resting NK cells constitutively express CD44 that is in an inactive form that does not bind to hyaluronan (HA), the principal known ligand for CD44. After infection of mice with the intracellular parasite Toxoplasma gondii, however, a population of NK cells that expressed activated CD44 emerged. To determine how expression and activation of CD44 by resting NK cells were regulated, the role of cytokines in these events was assessed. These studies revealed that whereas stimulation of resting NK cells with interleukin-12 (IL-12) or IL-18 caused increased expression of CD44, only IL-2 or IL-15 led to the upregulation and activation of CD44. The cytokine-induced upregulation and activation of CD44 was independent of NK cell proliferation. To determine the functional consequences of CD44 activation, the effects of low molecular weight HA (LMWHA) on the production of interferon-gamma (IFN-gamma) by IL-2-activated NK cells were assessed. These studies showed that HA alone had little effect on the production of IFN-gamma, but when used in combination with IL-2, IL-12, or IL-18, LMWHA was a potent enhancer of IFN-gamma production. Together, these studies indicate an important role for proinflammatory cytokines in the activation of CD44 on NK cells and identify a novel pathway to enhance the ability of activated NK cells to produce IFN-gamma.