Lymphocyte activation remains an area of intense interest to immunologists and cell biologists and the dynamics of expression of surface molecules during the process are widely studied. The CD69 C-type lectin is reportedly the earliest activation antigen on lymphocytes and can be detected within hours of mitogenic stimulation. Recently reports have described differential activation dynamics with respect to different antigenic or mitogenic stimuli. This study has investigated the dynamics of CD69 expression over time after mitogenic, allogeneic, cytokine and target cell mediated activation of T-cell and NK cell subsets. It is apparent that the dynamics of CD69 expression differ with respect to the cell type and the method of stimulation. Mitogenic stimulation resulted in the most rapid expression of CD69 on both T- and NK cells while alloantigen stimulation induced a far slower response. Target cell stimulation of NK cells gave paradoxical results in that the CD69 + ve subset increased as a proportion of the total NK cells but did not increase in number. This was due to the selective binding of CD69 - ve NK cells to the target cells and their subsequent loss from the lymphoid gate. We confirmed this by showing that CD69 + ve NK cells do not lyse K562 target cells. This observation demonstrates the caution needed in the analysis of flow cytometric data based solely upon relative proportions of cells within discrete subsets.