Human natural killer (NK) cells (CD56+ CD3−) represent crucial components of the innate immune system especially against viral infections and because their activation can modulate the outcome of the adaptive immune response. NKT cells (CD56+CD3+), a lymphocyte T population characterized by expression of surface markers of NK cells, are known to be abundant in the liver and their activation could be associated with hepatic injury. Using three‐color flow cytometry to measure surface receptors and intracellular cytokines, we have explored early activation signals and cytokine production in NK and NKT cells within a group of hepatitis B vaccinated and non‐vaccinated individuals. A specific increase of the CD56 bright cell population, the activation receptor CD69 and IFN‐γ, was observed in NK cells following incubation with recombinant HBsAg in responders to vaccination. Comparable results were observed in NKT cells showing an increment of CD69, CD25, IL‐2 and IFN‐γ expression in responder subjects. These parameters were statistically diminished in non‐responder individuals (p<0.05) in both groups of cells. These results demonstrate a diminished activation of these cells in non‐responders to the vaccine, suggesting that NK and NKT cells play an important role in the immune response following hepatitis B vaccination.