Environmental stress, including heavy metal stress, can cause oxidative damage to plants. Up-regulation of the antioxidant defense system induced by proline and glycinebetaine (betaine) alleviates the damaging effects of oxidative stress in plants. Here, we investigated the protective effects of exogenously applied proline and betaine on growth, accumulation of proline and betaine, lipid peroxidation and activity of antioxidant enzymes in cultured tobacco Bright Yellow-2 (BY-2) cells exposed to cadmium (Cd) stress. Cadmium stress (at 100 microM Cd) caused a significant inhibition of the growth of BY-2 cells, and both proline and betaine significantly mitigated this inhibition. In addition, the mitigating effect of proline was more pronounced than that of betaine. Cadmium stress leads to an accumulation of Cd and endogenous proline in cultured cells, increased lipid peroxidation and peroxidase (POX) activity, and decreased activity of superoxide dismutase (SOD) and catalase (CAT). Exogenous application of proline resulted in a decrease in lipid peroxidation and an increase in SOD and CAT activities without reducing Cd contents under Cd stress, while application of betaine resulted in a decrease in lipid peroxidation and an increase in CAT activity with reducing Cd accumulation. Furthermore, exogenous proline and betaine intensified the accumulation of proline and betaine in Cd-stressed BY-2 cells, respectively. The present study suggests that proline and betaine confer tolerance to Cd stress in tobacco BY-2 cells by different mechanisms.